Antibodies directed against interleukin 36 receptor (IL-36R)

ABSTRACT

The invention relates to an isolated immunoglobulin heavy chain polypeptide and an isolated immunoglobulin light chain polypeptide that bind to a protein encoded by the interleukin 36 receptor (IL-36R). The invention provides an IL-36R-binding agent that comprises the aforementioned immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide. The invention also provides related vectors, compositions, and methods of using the IL-36R-binding agent to treat a disorder or disease that is responsive to IL-36R inhibition, such as cancer, an infectious disease, or an autoimmune disease.

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 70,258 Byte ASCII (Text) file named“723558_ST25.TXT,” created on Apr. 13, 2016.

BACKGROUND OF THE INVENTION

The interleukin 36 (IL-36) cytokines IL-36α, IL-36β, and IL-36γ(formerly IL-1F6, IL-1F8, and IL-1F9) are interleukin-1 (IL-1) familymembers that bind to the IL-36 receptor (IL-36R) (formerly IL-1Rrp2 orIL-1RL2) and use IL-1 receptor accessory protein (IL-1RAcP) as acoreceptor to stimulate intracellular signals similar to those inducedby IL-1 (Towne et al., J. Biol. Chem., 279(14): 13677-13688 (2004)).IL-1F5 is an IL-1 family member that has been shown to act as anantagonist of IL-36R, and is now referred to as IL-36Ra (Dinarello etal., Nat. Immunol., 11(11): 973 (2010)).

IL-36α, IL-36β, and IL-36γ are highly expressed in several tissues,including internal epithelial tissues that have been exposed topathogens, and in skin. Expression of IL-36Ra and IL-36α issignificantly up-regulated in IL-1β/TNF-α-stimulated humankeratinocytes, and IL-36Rα and IL-36γ mRNAs are overexpressed inpsoriasis skin lesions. Elevated IL-36α mRNA and protein expression alsohave been observed in chronic kidney disease (Ichii et al., Lab Invest.,90(3): 459-475 (2010)). Both murine bone marrow-derived dendritic cells(BMDCs) and CD4+ T lymphocytes constitutively express IL-36R and responddirectly to IL-36α, IL-36β, and IL-36γ by producing proinflammatorycytokines (e.g., IL-12, IL-1β, IL-6, TNF-α, and IL-23) inducing a morepotent stimulatory effect than other IL-1 cytokines (Vigne et al.,Blood, 118(22): 5813-5823 (2011)).

Transgenic mice overexpressing IL-36α in keratinocytes exhibit atransient inflammatory skin disorder at birth that renders mice highlysusceptible to a 12-O-tetradecanoylphorbol 13-acetate-induced skinpathology resembling human psoriasis (Blumberg et al., J. Exp. Med.,204(11): 2603-2614 (2007); and Blumberg et al., J. Immunol.,185(7):4354-4362 (2010)). Furthermore, IL-36R-deficient mice areprotected from imiquimod-induced psoriasiform dermatitis (Tortola etal., J. Clin. Invest., 122(11): 3965-3976 (2012)). These resultsstrongly suggest a role for IL-36 in certain inflammatory disorders ofthe skin.

IL-36 cytokines also have been implicated in certain severe forms ofpsoriasis, including pustular psoriasis, generalized pustular psoriasis(GPP), and palmo-plantar pustulosis (PPP)) (see, e.g., Town, J. E. andSims, J. E., Curr. Opin. Pharmacol., 12(4): 486-90 (2012); and Naik, H.B. and Cowen, E. W., Dermatol Clin., 31(3): 405-425 (2013)). Pustularpsoriasis is a rare form of psoriasis characterized by white pustulessurrounded by red skin. Generalized pustular psoriasis is a severe,systemic form of pustular psoriasis that has a high risk of fatality,while palmo-plantar pustulosis is a chronic form of pustular psoriasisthat affects the palms and soles of the feet. Current treatments forpustular psoriasis, GPP, and PPP include oral retinoids and topicalsteroids, but these treatments exhibit poor efficacy and severe sideeffects.

There is a need for antagonists of IL-36R (e.g., an antibody) that bindIL-36R with high affinity and effectively neutralize IL-36R activity.The invention provides such IL-36R-binding agents.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of Gln Val Gln Xaa1Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser CysLys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln AlaPro Gly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 SerThr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6Ser Ala Xaa7 Thr Ala Tyr Met Glu Leu Xaa8 Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Xaa9 Cys Thr Arg Ser Phe Tyr Thr Met Asp Tyr Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser (SEQ ID NO: 56), wherein (a) Xaa1 is leucine(Leu) or phenylalanine (Phe), (b) Xaa2 is valine (Val), methionine(Met), or leucine (Leu), (c) Xaa3 is arginine (Arg) or glycine (Gly),(d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala), (e) Xaa5 isarginine (Arg) or alanine (Ala), (f) Xaa6 is threonine (Thr) or lysine(Lys), (g) Xaa7 is serine (Ser) or asparagine (Asn), (h) Xaa8 is serine(Ser) or alanine (Ala), and (i) Xaa9 is tyrosine (Tyr) or phenylalanine(Phe).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises the amino acid sequence of Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Xaa1 Met Xaa2 Trp Val Arg GlnAla Pro Xaa3 Gln Gly Leu Glu Trp Met Gly Met Phe Xaa4 Pro Xaa5 Xaa6 Xaa7Val Thr Arg Leu Asn Gln Lys Phe Lys Asp Arg Val Thr Met Thr Arg Asp ThrSer Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal Tyr Tyr Cys Ala Arg Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 15), wherein (a)Xaa1 is tryptophan (Trp) or tyrosine (Tyr), (b) Xaa2 is histidine (His),asparagine (Asn), or tyrosine (Tyr), (c) Xaa3 is glycine (Gly) orarginine (Arg), (d) Xaa4 is aspartic acid (Asp), glutamic acid (Glu), orhistidine (His), (e) Xaa5 is serine (Ser), threonine (Thr), or tyrosine(Tyr), (f) Xaa6 is asparagine (Asn) or glycine (Gly), and (g) Xaa7 isserine (Ser), alanine (Ala), or aspartic acid (Asp).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of Xaa1 Xaa2 GlnXaa3 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu ThrCys Thr Val Xaa4 Xaa5 Tyr Ser Ile Thr Xaa6 Asp Phe Ala Trp Asn Trp IleArg Gln Xaa7 Pro Gly Xaa8 Xaa9 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr SerGly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa10 Xaa11Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala AspThr Ala Xaa12 Tyr Xaa13 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa14 (SEQ ID NO: 57), whereinXaa1 is glutamine (Gln) or aspartic acid (Asp); Xaa2 is valine (Val) orleucine (Leu); Xaa3 is leucine (Leu) or phenylalanine (Phe); Xaa4 isthreonine (Thr) or serine (Ser); Xaa5 is glycine (Gly) or arginine(Arg); Xaa6 serine (Ser) or alanine (Ala); Xaa7 is proline (Pro) orphenylalanine (Phe); Xaa8 is lysine (Lys) or asparagine (Asn); Xaa9 isglycine (Gly) or lysine (Lys); Xaa10 is serine (Ser) or threonine (Thr);Xaa11 is valine (Val) or arginine (Arg); Xaa12 is threonine (Thr) orvaline (Val); Xaa13 is tyrosine (Tyr) or phenylalanine (Phe); and Xaa14is alanine (Ala) or absent.

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises the amino acid sequence of SEQ ID NO: 33,SEQ ID NO: 34, or SEQ ID NO: 35.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of Asp Ile Val MetThr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile SerCys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Xaa1 Asn Thr Tyr Leu Tyr TrpXaa2 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Xaa3 Arg Met SerAsn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysMet Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu IleLys (SEQ ID NO: 36), wherein (a) Xaa1 is glycine (Gly) or alanine (Ala),(b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr), and (c) Xaa3 istyrosine (Tyr) or serine (Ser).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of Asp Ile Val MetThr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile SerCys Arg Ser Ser Lys Ser Leu Leu His Xaa1 Asn Xaa2 Ile Thr Tyr Phe TyrTrp Tyr Leu Xaa3 Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Gln Met SerAsn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysAla Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu IleLys (SEQ ID NO: 40), (a)Xaa1 is serine (Ser) or arginine (Arg), (b) Xaa2is glycine (Gly) or alanine (Ala), and (c) Xaa3 is glutamine (Gln) orhistidine (His).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile ThrCys Arg Ala Ser Gln Xaa1 Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Xaa2 Leu His Ser Gly ValPro Ser Arg Phe Ser Xaa3 Ser Gly Ser Gly Xaa4 Asp Xaa5 Thr Phe Thr IleSer Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His ThrLeu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Xaa6 Xaa7 (SEQID NO: 58), wherein (a) Xaa1 is aspartic acid (Asp) or tryptophan (Trp),(b) Xaa2 is arginine (Arg) or methionine (Met), (c) Xaa3 is glycine(Gly), serine (Ser) or proline (Pro), (d) Xaa4 is threonine (Thr) orasparagines (Asn), (e) Xaa5 is phenylalanine (Phe) or tyrosine (Tyr),(f) Xaa6 is arginine (Arg) or absent, and (g) Xaa7 is threonine (Thr) orabsent.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence of SEQ ID NO: 48,SEQ ID NO: 49, or SEQ ID NO: 50.

In addition, the invention provides isolated or purified nucleic acidsequences encoding the foregoing immunoglobulin polypeptides, vectorscomprising such nucleic acid sequences, IL-36R-binding agents comprisingthe foregoing immunoglobulin polypeptides, nucleic acid sequencesencoding such IL-36R-binding agents, vectors comprising such nucleicacid sequences, isolated cells comprising such vectors, compositionscomprising such IL-36R-binding agents or such vectors with apharmaceutically acceptable carrier, and methods of treating a disorderthat is responsive to IL-36R inhibition by administering effectiveamounts of such compositions to mammals.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1A is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with hIL-36γ.

FIG. 1B is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with hIL-36β.

FIG. 1C is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with hIL-36α.

FIG. 1D is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with 50 ng/mL hIL-36α.

FIG. 1E is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with 20 ng/mL hIL-36β.

FIG. 1F is a graph depicting the results of the HEK human IL-36R/IL-8luciferase reporter assay described in Example 1 upon stimulation ofcells with 600 ng/mL hIL-36γ.

FIG. 2A is a graph depicting the results of the HEK cynomolgusIL-36R/IL-8 luciferase reporter assay described in Example 1 uponstimulation of cells with 2 ug/mL cynolL-36α.

FIG. 2B is a graph depicting the results of the HEK cynomolgusIL-36R/IL-8 luciferase reporter assay described in Example 1 uponstimulation of cells with 10 ug/mL cynoIL-36β.

FIG. 2C is a graph depicting the results of the HEK cynomolgusIL-36R/IL-8 luciferase reporter assay described in Example 1 uponstimulation of cells with 300 ng/mL cynoIL-36γ.

FIG. 3A is a graph depicting experimental data which illustrate thecurve for the antibody designated APE5281 binding to human IL-36R asdetermined by the KINEXA™ assay described in Example 2.

FIG. 3B is a graph depicting experimental data which illustrate thecurve for the antibody designated APE6194 binding to human IL-36R asdetermined by the BIACORE™ assay described in Example 2.

FIG. 3C is a graph depicting experimental data which illustrate thecurve for the antibody designated APE7247 binding to human IL-36R asdetermined by the KINEXA™ assay described in Example 2.

FIG. 4A is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 10 ng/mLhIL-36α.

FIG. 4B is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 1 ng/mLhIL-36β.

FIG. 4C is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 100 ng/mLhIL-36γ.

FIG. 4D is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 10 ng/mLhIL-36α.

FIG. 4E is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 1 ng/mLhIL-36β.

FIG. 4F is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 100 ng/mLhIL-36γ.

FIG. 4G is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 10 ng/mLhIL-36α.

FIG. 4H is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 1 ng/mLhIL-36β.

FIG. 4I is a graph depicting the results of the IL-8 secretion assay inprimary human keratinocytes described in Example 3 using 100 ng/mLhIL-36γ.

FIG. 5A is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 50 ng/mLcyno IL-36α.

FIG. 5B is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 10 ng/mLcyno IL-36β.

FIG. 5C is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 250 ng/mLcyno IL-36γ.

FIG. 5D is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 50 ng/mLcyno IL-36α.

FIG. 5E is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 10 ng/mLcyno IL-36β.

FIG. 5F is a graph depicting the results of the IL-8 secretion assay inprimary cynomolgus keratinocytes described in Example 4 using 250 ng/mLcyno IL-36γ.

FIG. 6A is a graph depicting the results of the IL-8 secretion assay inprimary human monocytes described in Example 5 using 5 ng/mL of IL-36β.

FIG. 6B is a graph depicting the results of the IL-8 secretion assay inprimary human monocytes described in Example 5 using 500 ng/mL IL-36β.

FIG. 7A is a graph depicting the results of the IL-8 secretion assay inprimary human peripheral blood mononuclear cells (PBMCs) described inExample 6 using 10 ng/mL of IL-36α.

FIG. 7B is a graph depicting the results of the IL-8 secretion assay inprimary human peripheral blood mononuclear cells (PBMCs) described inExample 6 using 1 ng/mL IL-36β.

FIG. 7C is a graph depicting the results of the IL-8 secretion assay inprimary human peripheral blood mononuclear cells (PBMCs) described inExample 6 using 100 ng/mL IL-36γ.

FIG. 8A is a graph depicting the results of the antibody/antigencross-competition binding assay described in Example 8 as determined byBIACORE™ assay using APE5100 as primary antibody.

FIG. 8B is a graph depicting the results of the antibody/antigencross-competition binding assay described in Example 8 as determined byBIACORE™assay using APE6155 as primary antibody.

FIG. 9A is a graph depicting the results of the competitive bindingassay described in Example 9 using CHO-K cells stably co-expressinghuman IL-36R and human IL-1RAcP.

FIG. 9B is a graph depicting the results of the competitive bindingassay described in Example 9 using CHO-K cells stably co-expressingcynomolgus monkey IL-36R variant 1 and cynomolgus monkey IL-1RAcP.

FIG. 10A is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 2 /IL-8cells stimulated with 20 ng/mL cynoIL-36γ.

FIG. 10B is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 1/IL-8cells stimulated with 300 ng/mL cynoIL-36γ.

FIG. 10C is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 3/IL-8cells stimulated with 100 ng/mL cynoIL-36γ.

FIG. 10D is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 2/IL-8cells stimulated with 300 ng/mL cynoIL-36γ.

FIG. 10E is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 3/IL-8cells stimulated with 300 ng/mL cynoIL-36γ.

FIG. 10F is a graph depicting the results of the luciferase reporterassay described in Example 1 using HEK cynomolgus IL-36R variant 4/IL-8cells stimulated with 300 ng/mL cynoIL-36γ.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated immunoglobulin heavy chainpolypeptide and/or an isolated immunoglobulin light chain polypeptide,or a fragment (e.g., antigen-binding fragment) thereof. The term“immunoglobulin” or “antibody,” as used herein, refers to a protein thatis found in blood or other bodily fluids of vertebrates, which is usedby the immune system to identify and neutralize foreign objects, such asbacteria and viruses. The polypeptide is “isolated” in that it isremoved from its natural environment. In a preferred embodiment, animmunoglobulin or antibody is a protein that comprises at least onecomplementarity determining region (CDR). The CDRs form the“hypervariable region” of an antibody, which is responsible for antigenbinding (discussed further below). A whole immunoglobulin typicallyconsists of four polypeptides: two identical copies of a heavy (H) chainpolypeptide and two identical copies of a light (L) chain polypeptide.Each of the heavy chains contains one N-terminal variable (V_(H)) regionand three C-terminal constant (C_(H)1, C_(H)2, and C_(H)3) regions, andeach light chain contains one N-terminal variable (V_(L)) region and oneC-terminal constant (C_(L)) region. The light chains of antibodies canbe assigned to one of two distinct types, either kappa (κ) or lambda(λ), based upon the amino acid sequences of their constant domains. In atypical immunoglobulin, each light chain is linked to a heavy chain bydisulfide bonds, and the two heavy chains are linked to each other bydisulfide bonds. The light chain variable region is aligned with thevariable region of the heavy chain, and the light chain constant regionis aligned with the first constant region of the heavy chain. Theremaining constant regions of the heavy chains are aligned with eachother.

The variable regions of each pair of light and heavy chains form theantigen binding site of an antibody. The V_(H) and V_(L) regions havethe same general structure, with each region comprising four framework(FW or FR) regions. The term “framework region,” as used herein, refersto the relatively conserved amino acid sequences within the variableregion which are located between the hypervariable or complementarydetermining regions (CDRs). There are four framework regions in eachvariable domain, which are designated FR1, FR2, FR3, and FR4. Theframework regions form the β sheets that provide the structuralframework of the variable region (see, e.g., C. A. Janeway et al.(eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y.(2001)).

The framework regions are connected by three complementarity determiningregions (CDRs). As discussed above, the three CDRs, known as CDR1, CDR2,and CDR3, form the “hypervariable region” of an antibody, which isresponsible for antigen binding. The CDRs form loops connecting, and insome cases comprising part of, the beta-sheet structure formed by theframework regions. While the constant regions of the light and heavychains are not directly involved in binding of the antibody to anantigen, the constant regions can influence the orientation of thevariable regions. The constant regions also exhibit various effectorfunctions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells.

The isolated immunoglobulin heavy chain polypeptide and the isolatedimmunoglobulin light chain polypeptide of the invention desirably bindto the interleukin 36 receptor (IL-36R), formerly known as IL-1Rrp2.IL-36R is a receptor of the IL-1R family, and binds to the ligandsIL-36α (formerly IL-1F6), IL-36β (formerly IL-1F8), and IL-36γ (formerlyIL-1F9) (see, e.g., Vigne et al., Blood, 118(22): 5813-5823 (2011)).IL-36α, IL-36β, and IL-36γ are members of the IL-1 family of cytokinesand bind to IL-36R and use IL-1 receptor accessory protein (IL-1RAcP) asa coreceptor to stimulate intracellular signals similar to those inducedby IL-1 (Towne et al., J. Biol. Chem., 279(14): 13677-13688 (2004)).IL-36 cytokines and IL-36R are highly expressed by keratinocytes andother epithelial cell types, as well as dendritic cells and naive CD4+T-cells (Towne et al., supra; Vigne et al., Blood, 118(22): 5813-5823(2011); and Vigne et al., Blood, 120(17): 3478-3487 (2012))

The inventive isolated immunoglobulin heavy chain polypeptide and theinventive isolated immunoglobulin light chain polypeptide can form anagent that binds to IL-36R and another antigen, resulting in a “dualreactive” binding agent (e.g., a dual reactive antibody).

Certain other antibodies which bind to IL-36R, and components thereof,are known in the art (see, e.g., U.S. Patent Publication 2013/0236471).Anti-IL-36R antibodies also are commercially available from sources suchas, for example, Abcam (Cambridge, Mass.), and R&D Systems, Inc.(Minneapolis, Minn.).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises, consists of, or consists essentially of theamino acid sequence of Gln Val Gln Xaal Xaa2 Gln Ser Gly Ala Glu Val LysLys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe ThrSer Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa3 Leu Glu Trp MetGly Trp Ile Tyr Pro Gly Asp Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys GlyArg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Xaa7 Thr Ala Tyr Met Glu LeuXaa8 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa9 Cys Thr Arg Ser PheTyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ IDNO: 56), wherein (a) Xaa1 is leucine (Leu) or phenylalanine (Phe), (b)Xaa2 is valine (Val), methionine (Met), or leucine (Leu), (c) Xaa3 isarginine (Arg) or glycine (Gly), (d) Xaa4 is glycine (Gly), serine(Ser), or alanine (Ala), (e) Xaa5 is arginine (Arg) or alanine (Ala),(f) Xaa6 is threonine (Thr) or lysine (Lys), (g) Xaa7 is serine (Ser) orasparagine (Asn), (h) Xaa8 is serine (Ser) or alanine (Ala), and (i)Xaa9 is tyrosine (Tyr) or phenylalanine (Phe). In some embodiments, theisolated immunoglobulin heavy chain polypeptide comprises, consists of,or consists essentially of the amino acid sequence Gln Val Gln Xaa1 Xaa2Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys LysAla Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala ProGly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 Ser ThrLys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 SerAla Ser Thr Ala Tyr Met Glu Leu Xaa7 Ser Leu Arg Ser Glu Asp Thr Ala ValTyr Xaa8 Cys Thr Arg Ser Phe Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser (SEQ ID NO: 1), wherein (a) Xaa1 is leucine (Leu) orphenylalanine (Phe), (b) Xaa2 is valine (Val), methionine (Met), orleucine (Leu), (c) Xaa3 is arginine (Arg) or glycine (Gly), (d) Xaa4 isglycine (Gly), serine (Ser), or alanine (Ala), (e) Xaa5 is arginine(Arg) or alanine (Ala), (f) Xaa6 is threonine (Thr) or lysine (Lys), (g)Xaa7 is serine (Ser) or alanine (Ala), and (h) Xaa8 is tyrosine (Tyr) orphenylalanine (Phe).

The inventive heavy chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 56 or SEQID NO: 1 with any one of the aforementioned amino acid substitutions inany suitable combination. In one embodiment, the immunoglobulin heavychain polypeptide comprises, consists of, or consists essentially of anamino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ IDNO: 14.

The invention also provides an isolated immunoglobulin heavy chainpolypeptide that comprises, consists of, or consists essentially of theamino acid sequence Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys LysPro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr AsnTyr Xaa1 Met Xaa2 Trp Val Arg Gln Ala Pro Xaa3 Gln Gly Leu Glu Trp MetGly Met Phe Xaa4 Pro Xaa5 Xaa6 Xaa7 Val Thr Arg Leu Asn Gln Lys Phe LysAsp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Thr SerMet Ile Ile Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val SerSer (SEQ ID NO: 15), wherein (a) Xaa1 is tryptophan (Trp) or tyrosine(Tyr), (b) Xaa2 is histidine (His), asparagine (Asn), or tyrosine (Tyr),(c) Xaa3 is glycine (Gly) or arginine (Arg), (d) Xaa4 is aspartic acid(Asp), glutamic acid (Glu), or histidine (His), (e) Xaa5 is serine(Ser), threonine (Thr), or tyrosine (Tyr), (f) Xaa6 is asparagine (Asn)or glycine (Gly), and (g) Xaa7 is serine (Ser), alanine (Ala), oraspartic acid (Asp).

The inventive heavy chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 15 with oneof the aforementioned amino acid substitutions in any suitablecombination. In one embodiment, the immunoglobulin heavy chainpolypeptide comprises, consists of, or consists essentially of an aminoacid sequence of any one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, or SEQ ID NO: 24.

The invention also provides an isolated immunoglobulin light chainpolypeptide which comprises, consists of, or consists essentially of theamino acid sequence of Xaa1 Xaa2 Gln Xaa3 Gln Glu Ser Gly Pro Gly LeuVal Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Xaa4 Xaa5 Tyr SerIle Thr Xaa6 Asp Phe Ala Trp Asn Trp Ile Arg Gln Xaa7 Pro Gly Xaa8 Xaa9Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro SerLeu Lys Ser Arg Val Thr Ile Xaa10 Xaa11 Asp Thr Ser Lys Asn Gln Phe SerLeu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa12 Tyr Xaa13 Cys AlaIle Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser Xaa14 (SEQ ID NO: 57), wherein Xaa1 is glutamine (Gln) oraspartic acid (Asp); Xaa2 is valine (Val) or leucine (Leu); Xaa3 isleucine (Leu) or phenylalanine (Phe); Xaa4 is threonine (Thr) or serine(Ser); Xaa5 is glycine (Gly) or arginine (Arg); Xaa6 serine (Ser) oralanine (Ala); Xaa7 is proline (Pro) or phenylalanine (Phe); Xaa8 islysine (Lys) or asparagine (Asn); Xaa9 is glycine (Gly) or lysine (Lys);Xaa10 is serine (Ser) or threonine (Thr); Xaa11 is valine (Val) orarginine (Arg); Xaa12 is threonine (Thr) or valine (Val); Xaa13 istyrosine (Tyr) or phenylalanine (Phe); and Xaa14 is alanine (Ala) orabsent. In some embodiments, the isolated heavy chain immunoglobulinpolypeptide comprises, consists of, or consists essentially of the aminoacid sequence Xaa1 Val Gln Xaa2 Gln Glu Ser Gly Pro Gly Leu Val Lys ProSer Gln Thr Leu Ser Leu Thr Cys Thr Val Xaa3 Gly Tyr Ser Ile Thr Ser AspPhe Ala Trp Asn Trp Ile Arg Gln Xaa4 Pro Gly Xaa5 Xaa6 Leu Glu Trp IleGly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser ArgVal Thr Ile Xaa7 Xaa8 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu SerSer Val Thr Ala Ala Asp Thr Ala Val Tyr Xaa9 Cys Ala Ile Arg Gly Pro TyrSer Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO:25), wherein (a) Xaa1 is glutamine (Gln) or aspartic acid (Asp), (b)Xaa2 is leucine (Leu) or phenylalanine (Phe), (c) Xaa3 is threonine(Thr) or serine (Ser), (d) Xaa4 is proline (Pro) or phenylalanine (Phe),(e) Xaa5 is lysine (Lys) or asparagine (Asn), (f) Xaa6 is glycine (Gly)or lysine (Lys), (g) Xaa7 is serine (Ser) or threonine (Thr), (h) Xaa8is valine (Val) or arginine (Arg), and (i) Xaa9 is tyrosine (Tyr) orphenylalanine (Phe).

The inventive heavy chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 57 or SEQID NO: 25 with one or more of the aforementioned amino acidsubstitutions in any suitable combination.In one embodiment, theimmunoglobulin heavy chain polypeptide comprises, consists of, orconsists essentially of an amino acid sequence of any one of SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, orSEQ ID NO: 54.

In another embodiment, the invention provides an isolated immunoglobulinheavy chain polypeptide which comprises, consists of, or consistsessentially of the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 34,or SEQ ID NO: 35

When the inventive immunoglobulin heavy chain polypeptide consistsessentially of an amino acid sequence of any one of SEQ ID NO: 1-SEQ IDNO: 35, additional components can be included in the polypeptide that donot materially affect the polypeptide, e.g., by influencing affinity ofthe inventive heavy chain polypeptide to IL-36R. Examples of suchcomponents include, for example, protein moieties such as biotin thatfacilitate purification or isolation, passenger mutations, sequencesfree of problematic sites including free cysteines, additionalglycosylation sites, and high-likelihood deamidation or isomerizationsites.

When the inventive immunoglobulin heavy chain polypeptide consists of anamino acid sequence of any one of SEQ ID NO: 1-SEQ ID NO: 35, thepolypeptide does not comprise any additional components (i.e.,components that are not endogenous to the inventive immunoglobulin heavychain polypeptide).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 1-SEQ ID NO: 35. Nucleic acidor amino acid sequence “identity,” as described herein, can bedetermined by comparing a nucleic acid or amino acid sequence ofinterest to a reference nucleic acid or amino acid sequence. The percentidentity is the number of nucleotides or amino acid residues that arethe same (i.e., that are identical) as between the sequence of interestand the reference sequence divided by the length of the longest sequence(i.e., the length of either the sequence of interest or the referencesequence, whichever is longer). A number of mathematical algorithms forobtaining the optimal alignment and calculating identity between two ormore sequences are known and incorporated into a number of availablesoftware programs. Examples of such programs include CLUSTAL-W,T-Coffee, and ALIGN (for alignment of nucleic acid and amino acidsequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versionsthereof) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (forsequence alignment and sequence similarity searches). Sequence alignmentalgorithms also are disclosed in, for example, Altschul et al., J.Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl.Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds.,Biological Sequence Analysis: Probabalistic Models of Proteins andNucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding,Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic AcidsRes., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings,Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

In another embodiment, the invention provides an immunoglobulin lightchain polypeptide that comprises, consists of, or consists essentiallyof the amino acid sequence Asp Ile Val Met Thr Gln Ser Pro Leu Ser LeuPro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser LeuLeu His Ser Asn Xaa1 Asn Thr Tyr Leu Tyr Trp Xaa2 Leu Gln Lys Pro GlyGln Ser Pro Gln Leu Leu Ile Xaa3 Arg Met Ser Asn Leu Ala Ser Gly Val ProAsp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser ArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr ProPhe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys (SEQ ID NO: 36), wherein(a) Xaa1 is glycine (Gly) or alanine (Ala), (b) Xaa2 is phenylalanine(Phe) or tyrosine (Tyr), and (c) Xaa3 is tyrosine (Tyr) or serine (Ser).

The inventive light chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 36 with oneor more of the aforementioned amino acid substitutions in any suitablecombination. In one embodiment, the isolated immunoglobulin light chainpolypeptide comprises, consists of, or consists essentially of an aminoacid sequence of any one of SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:39.

The invention also provides an immunoglobulin light chain polypeptidethat comprises, consists of, or consists essentially of the amino acidsequence Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro GlyGln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Xaa1 AsnXaa2 Ile Thr Tyr Phe Tyr Trp Tyr Leu Xaa3 Lys Pro Gly Gln Pro Pro GlnLeu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala GluAsp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe GlyGly Gly Thr Lys Val Glu Ile Lys (SEQ ID NO: 40), (a) Xaa1 is serine(Ser) or arginine (Arg), (b) Xaa2 is glycine (Gly) or alanine (Ala), and(c) Xaa3 is glutamine (Gln) or histidine (His).

The inventive light chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 40 with oneor more of the aforementioned amino acid substitutions in any suitablecombination. In one embodiment, the isolated immunoglobulin light chainpolypeptide comprises, consists of, or consists essentially of an aminoacid sequence of any one of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,or SEQ ID NO: 44.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises, consists of, or consists essentially of theamino acid sequence of Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Xaa1 Ile AsnAsn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Tyr Thr Ser Xaa2 Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa3 SerGly Ser Gly Xaa4 Asp Xaa5 Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp Thr Phe Gly GlyGly Thr Lys Val Glu Ile Lys Xaa6 Xaa7 (SEQ ID NO: 58), wherein (a) Xaa1is aspartic acid (Asp) or tryptophan (Trp), (b) Xaa2 is arginine (Arg)or methionine (Met), (c) Xaa3 is glycine (Gly), serine (Ser) or proline(Pro), (d) Xaa4 is threonine (Thr) or asparagines (Asn), (e) Xaa5 isphenylalanine (Phe) or tyrosine (Tyr), (f) Xaa6 is arginine (Arg) orabsent, and (g) Xaa7 is threonine (Thr) or absent. In some embodiments,the immunoglobulin light chain polypeptide comprises, consists of, orconsists essentially of the amino acid sequence Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys ArgAla Ser Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro SerArg Phe Ser Xaa1 Ser Gly Ser Gly Thr Asp Xaa2 Thr Phe Thr Ile Ser SerLeu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu ProTrp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys (SEQ ID NO: 45), wherein(a) Xaa1 is serine (Ser) or proline (Pro), and (b) Xaa2 is phenylalanine(Phe) or tyrosine (Tyr).

The inventive light chain polypeptide can comprise, consist of, orconsist essentially of the amino acid sequence of SEQ ID NO: 58 or SEQID NO: 45 with one or more of the aforementioned amino acidsubstitutions in any suitable combination. In one embodiment, theisolated immunoglobulin light chain polypeptide comprises, consists of,or consists essentially of an amino acid sequence of any one of SEQ IDNO: 46, SEQ ID NO: 47, or SEQ ID NO: 55.

In another embodiment, the invention provides an isolated immunoglobulinlight chain polypeptide which comprises, consists of, or consistsessentially of the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 49,or SEQ ID NO: 50

When the inventive immunoglobulin light chain polypeptide consistsessentially of an amino acid sequence of any one of SEQ ID NO: 36-SEQ IDNO: 50, additional components can be included in the polypeptide that donot materially affect the polypeptide, such as those described herein.When the inventive immunoglobulin light chain polypeptide consists of anamino acid sequence of any one of SEQ ID NO: 36-SEQ ID NO: 50, thepolypeptide does not comprise any additional components (i.e.,components that are not endogenous to the inventive immunoglobulin lightchain polypeptide).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 36-SEQ ID NO: 50. Nucleic acidor amino acid sequence “identity” can be determined using the methodsdescribed herein.

One or more amino acids of the aforementioned immunoglobulin heavy chainpolypeptides and/or light chain polypeptides can be replaced orsubstituted with a different amino acid. An amino acid “replacement” or“substitution” refers to the replacement of one amino acid at a givenposition or residue by another amino acid at the same position orresidue within a polypeptide sequence.

Amino acids are broadly grouped as “aromatic” or “aliphatic.” Anaromatic amino acid includes an aromatic ring. Examples of “aromatic”amino acids include histidine (H or His), phenylalanine (F or Phe),tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acidsare broadly grouped as “aliphatic.” Examples of “aliphatic” amino acidsinclude glycine (G or Gly), alanine (A or Ala), valine (V or Val),leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine(S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P orPro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (Nor Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R orArg).

Aliphatic amino acids may be sub-divided into four sub-groups. The“large aliphatic non-polar sub-group” consists of valine, leucine, andisoleucine. The “aliphatic slightly-polar sub-group” consists ofmethionine, serine, threonine, and cysteine. The “aliphaticpolar/charged sub-group” consists of glutamic acid, aspartic acid,asparagine, glutamine, lysine, and arginine. The “small-residuesub-group” consists of glycine and alanine. The group of charged/polaramino acids may be sub-divided into three sub-groups: the“positively-charged sub-group” consisting of lysine and arginine, the“negatively-charged sub-group” consisting of glutamic acid and asparticacid, and the “polar sub-group” consisting of asparagine and glutamine.

Aromatic amino acids may be sub-divided into two sub-groups: the“nitrogen ring sub-group” consisting of histidine and tryptophan and the“phenyl sub-group” consisting of phenylalanine and tyrosine.

The amino acid replacement or substitution can be conservative,semi-conservative, or non-conservative. The phrase “conservative aminoacid substitution” or “conservative mutation” refers to the replacementof one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz andSchirmer, Principles of Protein Structure, Springer-Verlag, New York(1979)). According to such analyses, groups of amino acids may bedefined where amino acids within a group exchange preferentially witheach other, and therefore resemble each other most in their impact onthe overall protein structure (Schulz and Schirmer, supra).

Examples of conservative amino acid substitutions include substitutionsof amino acids within the sub-groups described above, for example,lysine for arginine and vice versa such that a positive charge may bemaintained, glutamic acid for aspartic acid and vice versa such that anegative charge may be maintained, serine for threonine such that a free—OH can be maintained, and glutamine for asparagine such that a free—NH₂ can be maintained.

“Semi-conservative mutations” include amino acid substitutions of aminoacids within the same groups listed above, but not within the samesub-group. For example, the substitution of aspartic acid forasparagine, or asparagine for lysine, involves amino acids within thesame group, but different sub-groups. “Non-conservative mutations”involve amino acid substitutions between different groups, for example,lysine for tryptophan, or phenylalanine for serine, etc.

In addition, one or more amino acids can be inserted into theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides. Any number of any suitable amino acids can beinserted into the amino acid sequence of the immunoglobulin heavy chainpolypeptide and/or light chain polypeptide. In this respect, at leastone amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids),but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 orless amino acids), can be inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids) are inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.In this respect, the amino acid(s) can be inserted into any one of theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides in any suitable location. Preferably, the aminoacid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.

The inventive isolated immunoglobulin heavy chain polypeptide and lightchain polypeptides are not limited to polypeptides comprising thespecific amino acid sequences described herein. Indeed, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that competeswith the inventive immunoglobulin heavy chain polypeptide or light chainpolypeptide for binding to IL-36R. In this respect, for example, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that binds to thesame epitope of IL-36R recognized by the heavy and light chainpolypeptides described herein. Antibody competition can be assayed usingroutine peptide competition assays which utilize ELISA, Western blot, orimmunohistochemistry methods (see, e.g., U.S. Pat. Nos. 4,828,981 and8,568,992; and Braitbard et al., Proteome Sci., 4: 12 (2006)).

The invention provides an IL-36R-binding agent comprising, consistingessentially of, or consisting of one or more of the inventive isolatedamino acid sequences described herein. By “IL-36R-binding agent” ismeant a molecule, preferably a proteinaceous molecule, which bindsspecifically to the IL-36R protein. Preferably, the IL-36R-binding agentis an antibody or a fragment (e.g., antigen-binding fragment) thereof.The IL-36R-binding agent of the invention comprises, consistsessentially of, or consists of the inventive immunoglobulin heavy chainpolypeptide and/or the inventive immunoglobulin light chain polypeptide.In one embodiment, the IL-36R-binding agent comprises, consistsessentially of, or consists of the inventive immunoglobulin heavy chainpolypeptide or the inventive immunoglobulin light chain polypeptide. Inanother embodiment, the IL-36R-binding agent comprises, consistsessentially of, or consists of the inventive immunoglobulin heavy chainpolypeptide and the inventive immunoglobulin light chain polypeptide.

Any amino acid residue of the inventive immunoglobulin heavy chainpolypeptide and/or the inventive immunoglobulin light chain polypeptidecan be replaced, in any combination, with a different amino acidresidue, or can be deleted or inserted, so long as the biologicalactivity of the IL-36R-binding agent is not materially diminished (e.g.,enhanced or improved) as a result of the amino acid replacements,insertions, and/or deletions.

The “biological activity” of an IL-36R-binding agent refers to, forexample, binding affinity for a particular IL-36R epitope,neutralization or inhibition of IL-36R binding to its receptor(s),neutralization or inhibition of IL-36R activity in vivo (e.g., IC₅₀),pharmacokinetics, and cross-reactivity (e.g., with non-human homologs ororthologs of the IL-36R protein, or with other proteins or tissues). Incertain embodiments the inventive interleukin 36 receptor(IL-36R)-binding agent desirably exhibits one or more of the followingbiological activities: (a) inhibits the interaction between IL-36R andIL-36α, IL-36β, and/or IL-36γ, (b) inhibits intracellular signalingmediated by IL-36R, and/or (c) cross-reacts with and inhibits theactivity of human and non-human primate (e.g., cynomolgus) IL-36R. Otherbiological properties or characteristics of an antigen-binding agentrecognized in the art include, for example, avidity, selectivity,solubility, folding, immunotoxicity, expression, and formulation. Theaforementioned properties or characteristics can be observed, measured,and/or assessed using standard techniques including, but not limited to,ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™),or KINEXA™, in vitro or in vivo neutralization assays, receptor-ligandbinding assays, cytokine or growth factor production and/or secretionassays, and signal transduction and immunohistochemistry assays.

The terms “inhibit” or “neutralize,” as used herein with respect to theactivity of a IL-36R-binding agent, refer to the ability tosubstantially antagonize, prohibit, prevent, restrain, slow, disrupt,alter, eliminate, stop, or reverse the progression or severity of, forexample, the biological activity of IL-36R, or a disease or conditionassociated with IL-36R. The IL-36R-binding agent of the inventionpreferably inhibits or neutralizes the activity of IL-36R by at leastabout 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 95%, about 100%, or a range defined by any two ofthe foregoing values.

The IL-36R-binding agent of the invention can be a whole antibody, asdescribed herein, or an antibody fragment. The terms “fragment of anantibody,” “antibody fragment,” and “functional fragment of an antibody”are used interchangeably herein to mean one or more fragments of anantibody that retain the ability to specifically bind to an antigen(see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129(2005)). The IL-36R-binding agent can contain any IL-36R-bindingantibody fragment. The antibody fragment desirably comprises, forexample, one or more CDRs, the variable region (or portions thereof),the constant region (or portions thereof), or combinations thereof.Examples of antibody fragments include, but are not limited to, (i) aFab fragment, which is a monovalent fragment consisting of the V_(L),V_(H), C_(L), and CH₁ domains, (ii) a F(ab′)₂ fragment, which is abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, (iii) a Fv fragment consisting of the V_(L)and V_(H) domains of a single arm of an antibody, (iv) a Fab′ fragment,which results from breaking the disulfide bridge of an F(ab′)₂ fragmentusing mild reducing conditions, (v) a disulfide-stabilized Fv fragment(dsFv), and (vi) a domain antibody (dAb), which is an antibody singlevariable region domain (VH or VL) polypeptide that specifically bindsantigen.

In embodiments where the IL-36R-binding agent comprises a fragment ofthe immunoglobulin heavy chain or light chain polypeptide, the fragmentcan be of any size so long as the fragment binds to, and preferablyinhibits the activity of, IL-36R. In this respect, a fragment of theimmunoglobulin heavy chain polypeptide desirably comprises between about5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or a range defined by any two of the foregoing values) amino acids.Similarly, a fragment of the immunoglobulin light chain polypeptidedesirably comprises between about 5 and 18 (e.g., about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of theforegoing values) amino acids.

When the IL-36R-binding agent is an antibody or antibody fragment, theantibody or antibody fragment desirably comprises a heavy chain constantregion (F_(c)) of any suitable class. Preferably, the antibody orantibody fragment comprises a heavy chain constant region that is basedupon wild-type IgG1, IgG2, or IgG4 antibodies, or variants thereof. Itwill be appreciated that each antibody class, or isotype, engages adistinct set of effector mechanisms for disposing of or neutralizingantigen once recognized. As such, in some embodiments, when theIL-36R-binding agent is an antibody or antibody fragment, it can exhibitone or more effector functions, such as participation inantibody-dependent complement-mediated lysis or antibody-dependentcellular toxicity via interactions with effector molecules and cells(e.g., activation of the complement system).

The IL-36R-binding agent also can be a single chain antibody fragment.Examples of single chain antibody fragments include, but are not limitedto, (i) a single chain Fv (scFv), which is a monovalent moleculeconsisting of the two domains of the Fv fragment (i.e., V_(L) and V_(H))joined by a synthetic linker which enables the two domains to besynthesized as a single polypeptide chain (see, e.g., Bird et al.,Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA,85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778(1998)) and (ii) a diabody, which is a dimer of polypeptide chains,wherein each polypeptide chain comprises a V_(H) connected to a V_(L) bya peptide linker that is too short to allow pairing between the V_(H)and V_(L) on the same polypeptide chain, thereby driving the pairingbetween the complementary domains on different V_(H)-V_(L) polypeptidechains to generate a dimeric molecule having two functional antigenbinding sites. Antibody fragments are known in the art and are describedin more detail in, e.g., U.S. Patent Application Publication2009/0093024 A1.

The IL-36R-binding agent also can be an intrabody or fragment thereof.An intrabody is an antibody which is expressed and which functionsintracellularly. Intrabodies typically lack disulfide bonds and arecapable of modulating the expression or activity of target genes throughtheir specific binding activity. Intrabodies include single domainfragments such as isolated V_(H) and V_(L) domains and scFvs. Anintrabody can include sub-cellular trafficking signals attached to the Nor C terminus of the intrabody to allow expression at highconcentrations in the sub-cellular compartments where a target proteinis located. Upon interaction with a target gene, an intrabody modulatestarget protein function and/or achieves phenotypic/functional knockoutby mechanisms such as accelerating target protein degradation andsequestering the target protein in a non-physiological sub-cellularcompartment. Other mechanisms of intrabody-mediated gene inactivationcan depend on the epitope to which the intrabody is directed, such asbinding to the catalytic site on a target protein or to epitopes thatare involved in protein-protein, protein-DNA, or protein-RNAinteractions.

The IL-36R-binding agent also can be an antibody conjugate. In thisrespect, the IL-36R-binding agent can be a conjugate of (1) an antibody,an alternative scaffold, or fragments thereof, and (2) a protein ornon-protein moiety comprising the IL-36R-binding agent. For example, theIL-36R-binding agent can be all or part of an antibody conjugated to apeptide, a fluorescent molecule, or a chemotherapeutic agent.

The IL-36R-binding agent can be, or can be obtained from, a humanantibody, a non-human antibody, or a chimeric antibody. A “chimeric”antibody is an antibody or fragment thereof comprising both human andnon-human regions. Preferably, the IL-36R-binding agent is a humanizedantibody. A “humanized” antibody is a monoclonal antibody comprising ahuman antibody scaffold and at least one CDR obtained or derived from anon-human antibody. Non-human antibodies include antibodies isolatedfrom any non-human animal, such as, for example, a rodent (e.g., a mouseor rat). A humanized antibody can comprise, one, two, or three CDRsobtained or derived from a non-human antibody. In one embodiment of theinvention, CDRH3 of the inventive IL-36R-binding agent is obtained orderived from a mouse monoclonal antibody, while the remaining variableregions and constant region of the inventive IL-36R-binding agent areobtained or derived from a human monoclonal antibody.

A human antibody, a non-human antibody, a chimeric antibody, or ahumanized antibody can be obtained by any means, including via in vitrosources (e.g., a hybridoma or a cell line producing an antibodyrecombinantly) and in vivo sources (e.g., rodents). Methods forgenerating antibodies are known in the art and are described in, forexample, Köler and Milstein, Eur. J. Immunol., 5: 511-519 (1976); Harlowand Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); andJaneway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, NewYork, N.Y. (2001)). In certain embodiments, a human antibody or achimeric antibody can be generated using a transgenic animal (e.g., amouse) wherein one or more endogenous immunoglobulin genes are replacedwith one or more human immunoglobulin genes. Examples of transgenic micewherein endogenous antibody genes are effectively replaced with humanantibody genes include, but are not limited to, the MedarexHUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see,e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg,Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanized antibody can begenerated using any suitable method known in the art (see, e.g., An, Z.(ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, JohnWiley & Sons, Inc., Hoboken, N.J. (2009)), including, e.g., grafting ofnon-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri etal., Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)). In one embodiment, a humanized antibody can be producedusing the methods described in, e.g., U.S. Patent ApplicationPublication 2011/0287485 A1.

In one embodiment, a CDR (e.g., CDR1, CDR2, or CDR3) or a variableregion of the immunoglobulin heavy chain polypeptide and/or theimmunoglobulin light chain polypeptide described herein can betransplanted (i.e., grafted) into another molecule, such as an antibodyor non-antibody polypeptide, using either protein chemistry orrecombinant DNA technology. In this regard, the invention provides anIL-36R-binding agent comprising at least one CDR of an immunoglobulinheavy chain and/or light chain polypeptide as described herein. TheIL-36R-binding agent can comprise one, two, or three CDRs of animmunoglobulin heavy chain and/or light chain variable region asdescribed herein.

In a preferred embodiment, the IL-36R-binding agent binds an epitope ofIL-36R which blocks the binding of IL-36R to any of its ligands (e.g.,IL-36α, IL-36β, and IL-36γ) and inhibits IL-36R-mediated signaling. Theinvention also provides an isolated or purified epitope of IL-36R whichblocks the binding of IL-36R to any of its ligands in an indirect orallosteric manner.

The invention also provides one or more isolated or purified nucleicacid sequences that encode the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide, andthe inventive IL-36R-binding agent.

The term “nucleic acid sequence” is intended to encompass a polymer ofDNA or RNA, i.e., a polynucleotide, which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “nucleic acid” and “polynucleotide” as usedherein refer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated and/or capped polynucleotides. Nucleic acids aretypically linked via phosphate bonds to form nucleic acid sequences orpolynucleotides, though many other linkages are known in the art (e.g.,phosphorothioates, boranophosphates, and the like).

The invention further provides a vector comprising one or more nucleicacid sequences encoding the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide,and/or the inventive IL-36R-binding agent. The vector can be, forexample, a plasmid, episome, cosmid, viral vector (e.g., retroviral oradenoviral), or phage. Suitable vectors and methods of vectorpreparation are well known in the art (see, e.g., Sambrook et al.,Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994)).

In addition to the nucleic acid sequence encoding the inventiveimmunoglobulin heavy polypeptide, the inventive immunoglobulin lightchain polypeptide, and/or the inventive IL-36R-binding agent, the vectorpreferably comprises expression control sequences, such as promoters,enhancers, polyadenylation signals, transcription terminators, signalpeptides (e.g., the osteonectin signal peptide), internal ribosome entrysites (IRES), and the like, that provide for the expression of thecoding sequence in a host cell. Exemplary expression control sequencesare known in the art and described in, for example, Goeddel, GeneExpression Technology: Methods in Enzymology, Vol. 185, Academic Press,San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, and the RSV promoter. Inducible promoters include, forexample, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), theEcdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.),LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERTtamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res.,27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No.7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144(2005)).

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences.

The vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allow cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/008796 and WO 1994/028143; Wigler et al., Proc. Natl. Acad. Sci.USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA,78: 1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1-14(1981); Santerre et al., Gene, 30: 147-156 (1984); Kent et al., Science,237: 901-903 (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy etal., Cell, 22: 817-823 (1980); and U.S. Pat. Nos. 5,122,464 and5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al., Gene Therapy,11: 1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

Other suitable vectors include integrating expression vectors, which mayrandomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system fromInvitrogen (Carlsbad, Calif.) (e.g., pcDNA™5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Life Technologies(Carlsbad, Calif.), UCOE from Millipore (Billerica, Mass.), and pCI orpFN10A (ACT) FLEXI™ from Promega (Madison, Wis.).

Viral vectors also can be used. Representative commercially availableviral expression vectors include, but are not limited to, theadenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, TheNetherlands), the lentiviral-based pLP1 from Invitrogen (Carlsbad,Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH fromStratagene (La Jolla, Calif.).

Nucleic acid sequences encoding the inventive amino acid sequences canbe provided to a cell on the same vector (i.e., in cis). Aunidirectional promoter can be used to control expression of eachnucleic acid sequence. In another embodiment, a combination ofbidirectional and unidirectional promoters can be used to controlexpression of multiple nucleic acid sequences. Nucleic acid sequencesencoding the inventive polypeptides alternatively can be provided to thepopulation of cells on separate vectors (i.e., in trans). Each of thenucleic acid sequences in each of the separate vectors can comprise thesame or different expression control sequences. The separate vectors canbe provided to cells simultaneously or sequentially.

The vector(s) comprising the nucleic acid(s) encoding the inventivepolypeptides can be introduced into a host cell that is capable ofexpressing the polypeptides encoded thereby, including any suitableprokaryotic or eukaryotic cell. As such, the invention provides anisolated cell comprising the inventive vector. Preferred host cells arethose that can be easily and reliably grown, have reasonably fast growthrates, have well characterized expression systems, and can betransformed or transfected easily and efficiently.

Examples of suitable prokaryotic cells include, but are not limited to,cells from the genera Bacillus (such as Bacillus subtilis and Bacillusbrevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces,Salmonella, and Erwinia. Particularly useful prokaryotic cells includethe various strains of Escherichia coli (e.g., K12, HB101 (ATCC No.33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

Preferably, the vector is introduced into a eukaryotic cell. Suitableeukaryotic cells are known in the art and include, for example, yeastcells, insect cells, and mammalian cells. Examples of suitable yeastcells include those from the genera Kluyveromyces, Pichia,Rhinosporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeastcells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Suitable insect cells are described in, for example, Kitts et al.,Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993).Preferred insect cells include Sf-9 and HIS (Invitrogen, Carlsbad,Calif.).

Preferably, mammalian cells are utilized in the invention. A number ofsuitable mammalian host cells are known in the art, and many areavailable from the American Type Culture Collection (ATCC, Manassas,Va.). Examples of suitable mammalian cells include, but are not limitedto, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells(Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), humanembryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3cells (ATCC No. CCL92). Other suitable mammalian cell lines are themonkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651),as well as the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Other suitable mammalian cell linesinclude, but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, and BHK or HaK hamster cell lines, all of which areavailable from the ATCC. Methods for selecting suitable mammalian hostcells and methods for transformation, culture, amplification, screening,and purification of cells are known in the art.

In one embodiment, the mammalian cell is a human cell. For example, themammalian cell can be a human lymphoid or lymphoid derived cell line,such as a cell line of pre-B lymphocyte origin. Examples of humanlymphoid cells lines include, without limitation, RAMOS (CRL-1596),Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al.,Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86),PER.C6 cells (Crucell Holland B.V., Leiden, The Netherlands), andderivatives thereof.

A nucleic acid sequence encoding the inventive amino acid sequence maybe introduced into a cell by “transfection,” “transformation,” or“transduction.” “Transfection,” “transformation,” or “transduction,” asused herein, refer to the introduction of one or more exogenouspolynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art and include, forexample, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J.(ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression Protocols, Humana Press (1991)); DEAE-dextran;electroporation; cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors canbe introduced into host cells, after growth of infectious particles insuitable packaging cells, many of which are commercially available.

The invention provides a composition comprising an effective amount ofthe inventive immunoglobulin heavy chain polypeptide, the inventiveimmunoglobulin light chain polypeptide, the inventive IL-36R-bindingagent, the inventive nucleic acid sequence encoding any of theforegoing, or the inventive vector comprising the inventive nucleic acidsequence. Preferably, the composition is a pharmaceutically acceptable(e.g., physiologically acceptable) composition, which comprises acarrier, preferably a pharmaceutically acceptable (e.g., physiologicallyacceptable) carrier, and the inventive amino acid sequences,IL-36R-binding agent, or vector. Any suitable carrier can be used withinthe context of the invention, and such carriers are well known in theart. The choice of carrier will be determined, in part, by theparticular site to which the composition may be administered and theparticular method used to administer the composition. The compositionoptionally can be sterile. The composition can be frozen or lyophilizedfor storage and reconstituted in a suitable sterile carrier prior touse. The compositions can be generated in accordance with conventionaltechniques described in, e.g., Remington: The Science and Practice ofPharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa.(2001).

The invention further provides a method of treating a disorder in amammal that is responsive to IL-36R inhibition or neutralization. Themethod comprises administering the aforementioned composition to amammal having a disorder that is responsive to IL-36R inhibition orneutralization, whereupon the disorder is treated in the mammal. Adisorder that is “responsive to IL-36R inhibition” or “responsive toIL-36R neutralization” refers to any disease or disorder in which adecrease in IL-36R levels or activity has a therapeutic benefit inmammals, preferably humans, or the improper expression (e.g.,overexpression) or increased activity of IL-36R causes or contributes tothe pathological effects of the disease or disorder. Disorders that areresponsive to IL-36R inhibition include, for example, inflammatorydiseases, autoimmune diseases, respiratory diseases, metabolicdisorders, and cancer.

Inflammatory disorders include, for example, allergic inflammation ofthe skin, lungs, and gastrointestinal tract, atopic dermatitis (alsoknown as atopic eczema), asthma (allergic and non-allergic),epithelial-mediated inflammation, fibrosis (e.g., idiopathic pulmonaryfibrosis, scleroderma, kidney fibrosis, and scarring), allergicrhinitis, food allergies (e.g., allergies to peanuts, eggs, dairy,shellfish, tree nuts, etc.), seasonal allergies, and other allergies.

The inventive method can be used to treat any type of autoimmune disease(i.e., as disease or disorder caused by immune system overactivity inwhich the body attacks and damages its own tissues), such as thosedescribed in, for example, MacKay I. R. and Rose N. R., eds., TheAutoimmune Diseases, Fifth Edition, Academic Press, Waltham, Mass.(2014). Examples of autoimmune diseases that can be treated by theinventive method include, but are not limited to, multiple sclerosis,asthma, type 1 diabetes mellitus, rheumatoid arthritis, scleroderma,Crohn's disease, psoriasis vulgaris (commonly referred to as psoriasis),pustular psoriasis, generalized pustular psoriasis (GPP), palmo-plantarpustulosis (PPP), inflammatory bowel disease, psoriatic arthritis,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus(SLE), ulcerative colitis, and ankylosing spondylitis. In a preferredembodiment, the inventive method is used to treat pustular psoriasis,generalized pustular psoriasis, palmo-plantar pustulosis (PPP), orpsoriasis vulgaris.

Pustular psoriasis is a rare form of psoriasis characterized by whitepustules surrounded by red skin. Generalized pustular psoriasis (GPP) isa life-threatening disease characterized by sudden, repeated episodes ofhigh-grade fever, generalized rash, and disseminated pustules, withhyperleukocytosis and elevated serum levels of C-reactive protein, whichcan be caused by a deficiency in the interleukin-36-receptor antagonist(interleukin-36Ra) (Marrakchi et al., N. Engl. J. Med., 365(7):620-628(2011)). GPP often presents in patients with existing or prior psoriasisvulgaris (PV); however, GPP can develop in patients without a history ofPV (Sugiura et al., J. Invest. Derm., 133: 2514-2521 (2013)).Palmo-plantar pustulosis is a chronic inflammatory skin diseasecharacterized by sterile pustules and red, scaly skin on the palms andsoles that considerably impairs the quality of life of affectedindividuals (de Waal, A. C. and van de Kerkhof, P. C. M., J.Dermatological Treatment, 22(2): 102-105 (2011)).

Examples of respiratory diseases that can be treated by the inventivemethod include, but are not limited to, asthma, cystic fibrosis,emphysema, chronic obstructive pulmonary disease (COPD), and acuterespiratory distress syndrome. Examples of metabolic disorders that canbe treated by the inventive method include, but are not limited to,obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease.

The inventive method can be used to treat any type of cancer known inthe art, including but not limited to, melanoma, renal cell carcinoma,lung cancer, bladder cancer, breast cancer, cervical cancer, coloncancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroidcancer, stomach cancer, salivary gland cancer, prostate cancer,pancreatic cancer, leukemia, lymphoma, and Merkel cell carcinoma (see,e.g., Bhatia et al., Curr. Oncol. Rep., 13(6): 488-497 (2011)).

Administration of a composition comprising the inventive immunoglobulinheavy chain polypeptide, the inventive immunoglobulin light chainpolypeptide, the inventive IL-36R-binding agent, the inventive nucleicacid sequence encoding any of the foregoing, or the inventive vectorcomprising the inventive nucleic acid sequence induces an immuneresponse in a mammal. An “immune response” can entail, for example,antibody production and/or the activation of immune effector cells(e.g., T-cells).

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. Preferably,the effect is therapeutic, i.e., the effect partially or completelycures a disease and/or adverse symptom attributable to the disease. Tothis end, the inventive method comprises administering a“therapeutically effective amount” of the IL-36R-binding agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the IL-36R-binding agent to elicit adesired response in the individual. For example, a therapeuticallyeffective amount of an IL-36R-binding agent of the invention is anamount which decreases IL-36R bioactivity in a human.

Alternatively, the pharmacologic and/or physiologic effect may beprophylactic, i.e., the effect completely or partially prevents adisease or symptom thereof. In this respect, the inventive methodcomprises administering a “prophylactically effective amount” of theIL-36R-binding agent. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve a desired prophylactic result (e.g., prevention of diseaseonset).

A typical dose can be, for example, in the range of 1 pg/kg to 20 mg/kgof animal or human body weight; however, doses below or above thisexemplary range are within the scope of the invention. The dailyparenteral dose can be about 0.00001 μg/kg to about 20 mg/kg of totalbody weight (e.g., about 0.001 μg/kg, about 0.1 μg/kg , about 1 μg/kg,about 5 μg/kg, about 10 μg/kg, about 100 μg/kg, about 500 μg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two ofthe foregoing values), preferably from about 0.1 μg/kg to about 10 mg/kgof total body weight (e.g., about 0.5 μg/kg, about 1 μg/kg, about 50μg/kg, about 150 μg/kg, about 300 μg/kg, about 750 μg/kg, about 1.5mg/kg, about 5 mg/kg, or a range defined by any two of the foregoingvalues), more preferably from about 1 μg/kg to 5 mg/kg of total bodyweight (e.g., about 3 μg/kg, about 15 μg/kg, about 75 μg/kg, about 300μg/kg, about 900 μg/kg, about 2 mg/kg, about 4 mg/kg, or a range definedby any two of the foregoing values), and even more preferably from about0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about 2.5mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg,about 13 mg/kg, or a range defined by any two of the foregoing values).Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease symptoms occurs, oralternatively, the treatment can be continued for the lifetime of thepatient. However, other dosage regimens may be useful and are within thescope of the invention. The desired dosage can be delivered by a singlebolus administration of the composition, by multiple bolusadministrations of the composition, or by continuous infusionadministration of the composition.

The composition comprising an effective amount of the inventiveimmunoglobulin heavy chain polypeptide, the inventive immunoglobulinlight chain polypeptide, the inventive IL-36R-binding agent, theinventive nucleic acid sequence encoding any of the foregoing, or theinventive vector comprising the inventive nucleic acid sequence can beadministered to a mammal using standard administration techniques,including oral, intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. The composition preferably is suitable forparenteral administration. The term “parenteral,” as used herein,includes intravenous, intramuscular, subcutaneous, rectal, vaginal, andintraperitoneal administration. More preferably, the composition isadministered to a mammal using peripheral systemic delivery byintravenous, intraperitoneal, or subcutaneous injection.

Once administered to a mammal (e.g., a human), the biological activityof the inventive IL-36R-binding agent can be measured by any suitablemethod known in the art. For example, the biological activity can beassessed by determining the stability of a particular IL-36R-bindingagent. In one embodiment of the invention, the IL-36R-binding agent(e.g., an antibody) has an in vivo half life between about 30 minutesand 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25days, about 35 days, about 40 days, about 45 days, or a range defined byany two of the foregoing values). In another embodiment, theIL-36R-binding agent has an in vivo half life between about 2 hours and20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14days, about 17 days, about 19 days, or a range defined by any two of theforegoing values). In another embodiment, the IL-36R-binding agent hasan in vivo half life between about 10 days and about 40 days (e.g.,about 10 days, about 13 days, about 16 days, about 18 days, about 20days, about 23 days, about 26 days, about 29 days, about 30 days, about33 days, about 37 days, about 38 days, about 39 days, about 40 days, ora range defined by any two of the foregoing values).

The stability of the inventive IL-36R-binding agent can be measured interms of the transition mid-point value (T_(m)), which is thetemperature where 50% of the amino acid sequence is in its nativeconfirmation, and the other 50% is denatured. In general, the higher theT_(m), the more stable the protein. In one embodiment of the invention,the inventive IL-36R binding agent comprises a transition mid-pointvalue (T_(m)) in vitro of about 60-100° C. For example, the inventiveIL-36R binding agent can comprise a T_(m) in vitro of about 65-80° C.(e.g., 66° C., 68° C., 70° C., 71° C., 75° C., or 79° C.), about 80-90°C. (e.g., about 81° C., 85° C., or 89° C.), or about 90-100° C. (e.g.,about 91° C., about 95° C., or about 99° C.).

The stability of the inventive IL-36R binding agent can be measuredusing any other suitable assay known in the art, such as, for example,measuring serum half-life, differential scanning calorimetry (DSC),thermal shift assays, and pulse-chase assays. Other methods of measuringprotein stability in vivo and in vitro that can be used in the contextof the invention are described in, for example, Protein Stability andFolding, B. A. Shirley (ed.), Human Press, Totowa, N.J. (1995); ProteinStructure, Stability, and Interactions (Methods in Molecular Biology),Shiver J. W. (ed.), Humana Press, New York, N.Y. (2010); and Ignatova,Microb. Cell Fact., 4: 23 (2005).

The biological activity of a particular IL-36R-binding agent also can beassessed by determining its binding affinity to IL-36R or an epitopethereof. The term “affinity” refers to the equilibrium constant for thereversible binding of two agents and is expressed as the dissociationconstant (K_(D)). Affinity of a binding agent to a ligand, such asaffinity of an antibody for an epitope, can be, for example, from about1 picomolar (pM) to about 100 micromolar (μM) (e.g., from about 1picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1micromolar (μM), or from about 1 μM to about 100 μM). In one embodiment,the IL-36R-binding agent can bind to an IL-36R protein with a K_(D) lessthan or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001nM, or a range defined by any two of the foregoing values). In anotherembodiment, the IL-36R-binding agent can bind to IL-36R with a K_(D)less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of theforegoing values). Immunoglobulin affinity for an antigen or epitope ofinterest can be measured using any art-recognized assay. Such methodsinclude, for example, fluorescence activated cell sorting (FACS),separable beads (e.g., magnetic beads), surface plasmon resonance (SPR),solution phase competition (KINEXA™), antigen panning, competitivebinding assays, and/or ELISA (see, e.g., Janeway et al. (eds.),Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 2001).

The IL-36R-binding agent of the invention may be administered alone orin combination with other drugs. For example, the IL-36R-binding agentcan be administered in combination with other agents for the treatmentor prevention of the diseases disclosed herein, such as ananti-inflammatory agent including, for example, corticosteroids (e.g.,prednisone and fluticasone), non-steroidal anti-inflammatory drugs(NSAIDs) (e.g., aspirin, ibuprofen, and naproxen), biologics (e.g.,infliximab (REMICADE™), adalimumab (HUMIRA™), or etanercept (ENBREL™)),methotrexate (MTX), an oral retinoid (e.g. acitretin (SORIATANE™)), andtopical steroids.

In addition to therapeutic uses, the IL-36R-binding agent describedherein can be used in diagnostic or research applications. In thisrespect, the IL-36R-binding agent can be used in a method to diagnose adisorder or disease in which the improper expression (e.g.,overexpression) or increased activity of IL-36R causes or contributes tothe pathological effects of the disease or disorder. In a similarmanner, the IL-36R-binding agent can be used in an assay to monitorIL-36R protein levels in a subject being tested for a disease ordisorder that is responsive to IL-36R inhibition. Research applicationsinclude, for example, methods that utilize the IL-36R-binding agent anda label to detect an IL-36R protein in a sample, e.g., in a human bodyfluid or in a cell or tissue extract. The IL-36R-binding agent can beused with or without modification, such as covalent or non-covalentlabeling with a detectable moiety. For example, the detectable moietycan be a radioisotope (e.g., ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I) a fluorescentor chemiluminescent compound (e.g., fluorescein isothiocyanate,rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase,beta-galactosidase, or horseradish peroxidase), or prosthetic groups.Any method known in the art for separately conjugating anantigen-binding agent (e.g., an antibody) to a detectable moiety may beemployed in the context of the invention (see, e.g., Hunter et al.,Nature, 194: 495-496 (1962); David et al., Biochemistry, 13: 1014-1021(1974); Pain et al., J. Immunol. Meth., 40: 219-230 (1981); and Nygren,J. Histochem. and Cytochem., 30: 407-412 (1982)).

IL-36R protein levels can be measured using the inventive IL-36R-bindingagent by any suitable method known in the art. Such methods include, forexample, radioimmunoassay (RIA), and FACS. Normal or standard expressionvalues of IL-36R can be established using any suitable technique, e.g.,by combining a sample comprising, or suspected of comprising, IL-36Rwith an IL-36R-specific antibody under conditions suitable to form anantigen-antibody complex. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, and radioactive materials (see, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amountof IL-36R polypeptide expressed in a sample is then compared with astandard value.

The IL-36R-binding agent can be provided in a kit, i.e., a packagedcombination of reagents in predetermined amounts with instructions forperforming a diagnostic assay. If the IL-36R-binding agent is labeledwith an enzyme, the kit desirably includes substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides adetectable chromophore or fluorophore). In addition, other additives maybe included in the kit, such as stabilizers, buffers (e.g., a blockingbuffer or lysis buffer), and the like. The relative amounts of thevarious reagents can be varied to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders (typicallylyophilized), including excipients which on dissolution will provide areagent solution having the appropriate concentration.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides can form antibodies that bind toand block the signaling of human IL-36R in vitro.

HEK293T/17 cells (ATCC CRL-11268) were stably transfected with a plasmidconstruct encoding either human IL-36R (hIL-36R) or cynomolgus IL-36R(cynolL-36R) together with an IL-8 promoter (Promega Corp., Madison,Wis.), and a single cell clone was chosen for all subsequent assays.

HEK293 cells were plated at 3×10⁶ cells/flask onto a T75 culture flaskin 10 mL of DMEM+10% FBS and incubated overnight at 37° C. The nextmorning, 24 μl A FUGENE™ HD (Promega Corporation, Madison, Wis.) wasadded to 500 μl OPTI-MEM™ medium (Life Technologies, Carlsbad, Calif.)and incubated at room temperature for five minutes. DNA encoding IL-36R(2 μl) and DNA encoding the IL8 promoter (2 μl) were added to themixture and allowed to incubate for an additional 25 minutes at roomtemperature. Cynomolgus IL-36R allelic variation was examined by Sangersequencing, and four distinct allelic variants were identified withincynomolgus monkey populations. HEK cell lines expressing each cynomolgusIL-36R allelic variants were generated separately. For both human andcynomolgus monkey IL-8 reporter cell lines, the endogenously expressedHEK human IL1RAcP was utilized. This DNA/FUGENE™ mixture was gentlyadded to the cells drop-wise for transfection and incubated overnight at37° C. 24 hours post-transfection, cells were split and placed inhygromycin and puromycin containing DMEM+10% FBS for four weeks forselection. After 4 weeks, stabilized cells were plated at 1 cell/well ona 96-well clear bottom plates (5 plates/cell line). Single cell cloneswere screened for surface expression of IL-36R and expanded, with lowpassage number (i.e., 1-3) cells used for the assay described below.

HEK293-human IL36R/IL8 or HEK293-cynoIL36R/IL8 variant stable cell lineswere harvested with accutase and seeded with 0.06×10⁶ cells/well in 100μl DMEM+10% FBS on a 96-well clear-bottomed plate overnight at 37° C.,5% CO₂. The next morning, plates were inverted into the sink to removemedia and gently tapped on a paper towel to dry. Diluted antibodiescomprising various combinations of the inventive HC and LC polypeptides(see Table 1), IL-36Ra (R&D Systems, Minneapolis, Minn.), and a negativeisotype control antibody were prepared in DMEM+10% FBS (LifeTechnologies, Carlsbad, Calif.) to the desired concentrations bytwo-fold dilution series, immediately added to the wells (50 μl/well),and allowed to incubate for 20 minutes at 37° C., 5% CO₂.

TABLE 1 Antibody Designation HC SEQ ID NO: LC SEQ ID NO: APE3798 33 48APE4086 3 38 APE5125/APE5100 4 39 APE5216 5 39 APE5281 6 39APE5214/APE4881 7 39 APE5280 8 39 APE5257 9 39 APE5258/APE5076 10 39APE5212 11 39 APE5213/5066 12 39 APE5211 13 39 APE5217/APE5060 14 39APE3849 34 49 APE3850 16 41 APE5600 18 42 APE5598 19 42 APE5627 20 42APE6064 21 43 APE6060 22 43 APE6157 23 43 APE6155/APE6917 22 44 APE619424 44 APE3847 35 50 APE5713 27 47 APE6083 32 47 APE6903/APE7247 52 55APE6904 53 55 APE6907 54 55

Cells were subsequently stimulated with 50 μl of IL36α, IL36β, or IL36γligands (R&D Systems, Minneapolis, Minn.) and allowed to incubate for anadditional 24 hours at 37° C., 5% CO₂. EC₅₀s of each of the individualcytokines were determined empirically prior to the assay. Luciferaseactivity was determined by using STEADY-GLO™ Luciferase Assay System(Promega, Cat# E2520, Madison, Wis.). 100 μl of 1:1 mix of luciferaseassay substrate:buffer was added to each well, incubated for fiveminutes at room temperature, and transferred (150 ul) onto 96-well blackwalled, clear bottom plates. Plates were read on ENVISION™ Plate Reader(PerkinElmer, Waltham, Wash.) to determine luminescence (60-sec delay).Data was analyzed using GraphPad PRISM™ Software 5 (GraphPad, San Diego,Calif.).

The results of the IL-8 luciferase reporter assay against human and cynoIL-36R are shown in FIGS. 1A-1F (human IL-36R), FIGS. 2A-2C (cynoIL-36R), FIGS. 10A-10C (cyno IL-36R), and FIGS. 10D-10F (human IL-36R).The measured potencies (IC₅₀) of each of the tested antibodies are setforth in Tables 2 and 3.1 and 3.2.

TABLE 2 HEK human IL-36R IL-8 luciferase reporter assay EC50 (nM) EC50(nM) EC50 (nM) HC SEQ LC SEQ 50 ng/ml 20 ng/ml 200 ng/ml Antibody ID NO:ID NO: hIL-36α hIL-36β hIL-36γ Chimeric 1D9 33 48 0.267 0.093 (APE3798)Humanized 6 39 0.17 0.12 (HzD) 1D9 (APE5281) Chimeric 5D3 34 49 1.3 3.1(APE3849) Hzd 5D3 22 43 0.23 0.24 0.35 (APE6060) Hzd 5D3 22 44 0.23 0.220.40 (APE6155) Hzd 5D3 24 44 0.17 0.30 0.45 (APE6194) Chimeric 18D4 3550 4.2 3.6 (APE3847) Hzd 18D4 27 47 11 (APE5713) Hzd 18D4 52 55 .0660.114 0.104 (APE7247)

TABLE 3.1 HEK cyno IL-36R IL-8 luciferase reporter assay EC50 (nM) EC50(nM) EC50 (nM) HC SEQ LC SEQ 2 μg/ml 10 μg/ml 300 ng/ml Antibody ID NO:ID NO: cIL-36α cIL-36β cIL-36γ Hzd 5D3 22 43 0.067 0.17 0.29 (APE6060)Hzd 5D3 22 44 0.08 0.13 0.31 (APE6155) Hzd 5D3 24 44 0.073 0.19 0.31(APE6194) Chimeric 18D4 35 50 0.51 (APE3847) Hzd 18D4 52 55 0.24(APE7247)

TABLE 3.2 EC50 (nM) EC50 (nM) EC50 (nM) 300 ng/ml 300 ng/ml 300 ng/mlIL-36γ IL-36γ IL-36γ HC SEQ LC SEQ (Cyno IL-36R (Cyno IL-36R (CynoIL-36R Antibody ID NO: ID NO: variant 1) variant 2) variant 3) Hzd 18D452 55 0.079 0.065 0.42 (APE7247) Hzd 5D3 22 44 0.042 0.043 0.21(APE6155)

The results from this example demonstrate that the inventiveimmunoglobulin heavy chain (HC) and light chain (LC) polypeptides canform antibodies that bind to and inhibit signaling of human IL-36R invitro.

EXAMPLE 2

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides can form antibodies that bind tohuman IL-36R in vitro.

DNA samples encoding various immunoglobulin heavy chain (HC) and lightchain (LC) polypeptides as described herein were prepared by combiningthe following: maxi-prepped DNA (containing 6 μg HC plasmid and 6 μg LCplasmid), 1 ml OPTIMEM™ (Life Technologies, Carlsbad, Calif.), and 72 μlFUGENE™ HD Transfection Reagent (Promega, Fitchburg, Wis.). All reagentswere pre-warmed. Following thorough mixing and incubation for 25 minutesat room temperature, 1 ml of reagent/DNA mix was added to 8×10⁶HEK293-c18 cells (ATCC CRL-10852) in each T225 culture flask. 18 hoursprior to transfection, the cells were plated in T225 culture flasks with20 ml of DMEM (Life Technologies, Carlsbad, Calif.) with 10% FBS (LifeTechnologies, Carlsbad, Calif.) per flask and incubated at 37° C. in 5%CO₂ overnight. Following transfection, cells were returned to 37° C. in5% CO₂. The following day, the medium in each flask was exchanged with25 ml 293 Freestyle medium (Life Technologies, Carlsbad, Calif.), andcells were moved to an incubator at 8% CO₂. Antibody production wascarried out for 7-12 days. Supernatants were collected from each flask,spun down at 3000 rpm for 10 minutes, and sterile-filtered into freshtubes.

For antibody purification, approximately 20-30 ml of cell culturesupernatants containing the antibodies of interest were passed through agravity column packed with 1-2 ml MAB SELECT SURE™ LX resin (GEHealthcare, Waukesha, Wis.) pre-equilibrated with PBS buffer (11.9 mMphosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) (Fisher Bioreagents,Waltham, Mass.). The column was washed with five column volumes of PBSbuffer. Bound antibodies were eluted from the resin with 5-10 columnvolumes of 0.1 M glycine pH 3.0. The eluate containing the antibodieswas concentrated down to an antibody concentration of approximately0.1-2 mg/mL in Amicon Ultra 10K concentrators (Millipore, Billerica,Mass.), and buffer was exchanged three times against PBS buffer.Antibody concentration was determined on a Nanodrop 2000cspectrophotometer (Thermo Fisher Scientific, Waltham, Mass.), and puritywas assessed by SDS-PAGE analysis.

The binding affinities of various purified antibodies comprisingimmunoglobulin heavy chain (HC) and light chain (LC) polypeptidesdescribed herein were evaluated using BIACORE™ T200 (SapidyneInstruments, Boise, Id.) assays. BIACORE™ T200 evaluation software (GEHealthcare, Buckinghamshire, United Kingdom) is used to determineantibody-antigen binding kinetics and affinity. The extracellular domainof human IL-36Rwas immobilized at approximately 100 RU onto a CMS sensorchip (GE Healthcare, Waukesha, Wis.) using amine coupling chemistry.HBS-EP+buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Polysorbate,pH 7.6) (Teknova, Hollister, Calif.) was used to reconstitute eachantibody at various concentrations Each antibody concentration was theninjected for two to three minutes over immobilized antigen at a flowrate of 30 μL/min, and allowed to dissociate for 15 minutes. The surfacewas regenerated with 60 μL of 3 M MgCl₂ after each cycle. Associationand dissociation kinetic constants (kon and koff) were fit globallyusing a 1:1 binding model with mass transport with the BIACORE™ T200evaluation software in order to report on- and off-rates (ka and kd,respectively), as well as affinities (KD).

The binding affinities of various purified antibodies comprisingimmunoglobulin heavy chain (HC) and light chain (LC) polypeptidesdescribed herein also were evaluated using a KINEXA® 3000 assay(SapidyneInstruments, Boise, Id.) assays. KINEXA® technology measures the amountof unbound/free antibody molecule in solution phase after incubationwith varying concentrations of antigen. Measuring binding events in thesolution phase with micro beads for maximized surface area avoids masstransport limitations and mobility effects inherent to methods thatmeasure binding to a solid phase. For each experiment, 50 μg of solublehuman or cyno IL-36R extracellular domain was amine-coupled to 50 mg ofUltraLink Biosupport beads (Thermo Fisher Scientific, Waltham, Mass.). Aconstant concentration of antibody (sufficient to produce 0.8 V-1.2 V ofsignal) was incubated for a sufficient period of time to approach or toreach equilibrium (time of incubation varies for each antibody and isdependent on affinity) with titrated antigen in sample buffer (1× PBS,pH 7.4, 0.02% NaN₃, 0.1% BSA). Antibody-antigen solution was then flowedover antigen-coupled beads at a rate of 0.25 mL/min. Free antibodycaptured by beads was detected using ALEXA FLUOR™ 647-conjugatedAffiniPure Donkey Anti-Human IgG (H+L) (Jackson ImmunoResearch, WestGrove, Pa.) (500 ng/ml). The KD and/or ABC (active bindingconcentration) of antibody was obtained from non-linear regressionanalysis using a one-site homogeneous binding model in the KINEXA™ ProSoftware.

The resulting KD values of the BIACORE™ T200 and KINEXA® 3000 assaysassay are set forth in Table 4 and FIG. 3A (KinExA data for Humanized1D9), FIG. 3B (Biacore for 5D3 APE6194) and FIG. 3C (KinExA data forHumanized 18D4).

TABLE 4 BIACORE ™ BIACORE ™ KINEXA ™ KD human KD cyno KD humanDescription Antibody IL-36R IL-36R IL-36R 1D9 Humanized APE5281 77 pM126 pM  8 pM 5D3 Chimeric APE3850 35 pM 5D3 Humanized APE6060 50 pM 5D3Humanized APE6155 71 pM 169 pM 5D3 Humanized APE6194 22 pM 18D4Humanized APE7247 <20 pM  <20 pM 100 pM 18D4 Humanized APE6904  27 pM

These data demonstrate that antibodies comprising different combinationsof the inventive immunoglobulin HC and LC polypeptides described hereincan bind human IL-36R with high affinities.

EXAMPLE 3

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides can form antibodies that bind tohuman IL-36R in vitro and inhibit cell signaling and cytokine (e.g.,IL-8) release by human primary keratinocyte cells endogenouslyexpressing IL-36R.

Antibodies used in this assay were produced and purified as describedabove. Normal human epidermal kerotinocytes (NHEK) were purchased fromLonza Clonetics (cat #00192627). Cells were cultured and expanded usingrecommended culture medium (Lonza KBM Gold medium, cat #00192151 withLonza KGM Gold SingleQuot supplements, cat #0092152) in a 5% CO₂ 37° C.incubator. Cells were frozen in liquid nitrogen at passage 2 in multiplesingle use aliquots.

Passage 2 cells were thawed and diluted to a density of 100,000 cellsper ml in above recommended culture medium described above and 100 μlcells per well were plated in standard flat-bottom 96-well tissueculture plates for a final cell density of 10,000 cells per well.Outside wells are filled with 200 μl phosphate buffered saline per wellto avoid edge effects. Cells were cultured overnight in a 5% CO₂ 37° C.incubator to allow for adherence.

The following day antibodies were added at concentrations from 10 μg/mlor 1 μg/ml down to 0 by half-log dilutions in culture medium. After 30minutes, recombinant human IL-36 ligands were added at approximatelyEC₅₀ concentrations (previously determined empirically for each ligand)in culture medium. Antibody and ligand concentrations were made at 4× ofdesired final concentrations and 50 μl per well were added for a finaltotal volume in each well of 200 μl. Supernatants were removedapproximately 48 hours later following a three-minute centrifugation ofthe plates, transferred to clean plates, and either tested immediatelyor stored at −80° C. until further analysis.

Human IL-8 levels in the cell supernatants were assessed by ELISA usingR&D Systems DUO-SET™ ELISA kit (cat #DY208) following a standardprotocol provided by the manufacturer. Data were graphed and IC₅₀ valueswere calculated using GraphPad PRISM™ software.

The results of this assay are shown in Table 5 and FIGS. 4A-4I.

TABLE 5 EC50 (nM) EC50 (nM) EC50 (nM) HC SEQ LC SEQ 10 ng/ml 1 ng/ml 100ng/ml Antibody ID NO: ID NO: human IL-36α human IL-36β human IL-36γHumanized 6 39 0.047 0.053 0.04 (HzD) 1D9 (APE5281) Hzd 5D3 22 43 0.080.217 0.08 (APE6060) Hzd 5D3 22 44 0.125 0.227 0.093 (APE6155) Hzd 5D324 44 0.105 0.164 0.083 (APE6194) Hzd 18D4 52 55 0.142 0.336 0.074(APE7247)

The results of this example demonstrate that antibodies composed ofcombinations of HCs and LCs described herein inhibit inflammatorycytokine release (IL-8) from human primary keratinocytes expressingIL-36R and stimulated with cytokines IL-36α, IL-36β and IL-36γ in adose-dependent manner.

EXAMPLE 4

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides can form antibodies that bind tocynomolgus IL-36R (cyno IL-36R) in vitro and inhibit IL-36-dependentcell signaling and cytokine (e.g., IL-8) release by primary keratinocytecells endogenously expressing IL-36R.

Antibodies used in this assay were produced and purified as described inExample 3. Normal cynomolgus monkey epidermal kerotinocytes werepurchased from CellBiologics (Chicago, Ill.; cat #MK-6066K). Cells werecultured and expanded using recommended culture medium (CellBiologicsepithelial medium, cat #M6621 with CellBiologics epithelial cell mediumsupplements, cat #M6621-kit) in a 5% CO₂ 37° C. incubator. Cells werefrozen in liquid nitrogen at passage 2 in multiple single use aliquots.

Passage 2 cells were thawed and diluted to a density of 100,000 cellsper ml in culture medium described above, and 100 μl cells per well wereplated in standard flat-bottom 96-well tissue culture plates for final acell density of 10,000 cells per well. Outside wells were filled with200 μl PBS per well to avoid edge effects. Cells were cultured overnightin a 5% CO₂ 37° C. incubator to allow for adherence.

The following day antibodies were added at concentrations from 10 μg/mlor 1 μg/ml down to 0 by half-log dilutions in culture medium. After 30minutes, recombinant cynomolgus IL-36 ligands were added atapproximately EC₅₀ concentrations (previously determined empirically foreach ligand) in culture medium. Antibody and ligand concentrations weremade at 4× of desired final concentrations and 50 μl per well were addedfor a final total volume in each well of 200 μl. Supernatants wereremoved approximately 48 hours later following a three-minutecentrifugation of the plates, transferred to clean plates, and eithertested immediately or stored at −80° C. until further analysis.

Cynomolgus IL-8 levels in the cell supernatants were assessed by ELISAusing a eBioscience (San Diego, Calif.) monkey IL-8 platinum ELISA kit(cat #BMS640/3) following a standard protocol provided by themanufacturer. Data were graphed and IC₅₀ values were calculated usingGraphPad PRISM™ software.

The results of this assay are shown in Table 6 and FIGS. 5A-5F.

TABLE 6 EC50 (nM) EC50 (nM) EC50 (nM) HC SEQ LC SEQ 50 ng/ml 10 ng/ml250 ng/ml Antibody ID NO: ID NO: cyno IL-36α cyno IL-36β cyno IL-36γ Hzd5D3 22 43 1.4 1.4 1.0 (APE6060) Hzd 5D3 22 44 1.1 1.4 1.1 (APE6155) Hzd5D3 24 44 1.2 2.0 1.2 (APE6194) Hzd 18D4 52 55 2.3 1.8 5.6 (APE7247)

The results of this example demonstrate that antibodies composed ofcombinations of HCs and LCs described herein inhibit inflammatorycytokine release (IL-8) from cynomolgus primary keratinocytes expressingIL-36R and stimulated with cytokines IL-36α, IL-36β and IL-36γ in adose-dependent manner.

EXAMPLE 5

This example demonstrates the ability of antibodies composed of HCs andLCs described herein to block, in a dose-dependent manner, the humanIL-36-mediated release of IL-8 from human monocytes expressing theIL-36R.

A Leukocyte Reduction System unit processed from a donor whole bloodunit was obtained from the San Diego Blood Bank. Peripheral bloodmononuclear cells (PBMCs) were prepped by standard methods using Ficolldensity centrifugation separation (Sigma HISTOPAQUE™ cat #10771).Monocytes were isolated from PBMCs with human monocyte isolation kit II(Miltenyi Biotec, San Diego, Calif.; cat #130-091-153).

Monocytes were diluted to a density of 500,000 cells/ml in RPMI 1640medium containing 10% fetal bovine serum and penicillin/streptomycin,and 100 μl cells per well were plated in standard flat-bottom 96-welltissue culture plates for a final cell density of 50,000 per well.Outside wells were filled with 200 μl PBS per well to avoid edgeeffects. Plated cells were incubated for 2-3 hours in a 5% CO₂ 37° C.incubator to allow for recovery.

After approximately 2-3 hours of culture, antibodies were added atconcentrations from 10 μg/ml or 1 μg/ml down to 0 by half-log dilutionsin culture medium. After 30 minutes, recombinant human IL-36 ligandswere added at approximately EC₅₀ concentrations (previously determinedempirically for each ligand) in culture medium. Antibody and ligandconcentrations were made at 4× of desired final concentrations and 50 μlper well were added for a final total volume in each well of 200 μl.Supernatants were removed approximately 48 hours later following athree-minute centrifugation of the plates, transferred to clean plates,and either tested immediately or stored at −80° C. until furtheranalysis.

Human IL-8 levels in the cell supernatants were assessed by ELISA usingR&D Systems DUO-SET™ ELISA kit (cat #DY208) following a standardprotocol provided by the manufacturer. Data were graphed and IC₅₀ valueswere calculated using GraphPad PRISM™ software.

The results of these experiments are set forth in Table 7 and FIGS. 6Aand 6B.

TABLE 7 HC SEQ LC SEQ EC50 (nM) EC50 (nM) EC50 (nM) Antibody ID NO: IDNO: human IL-36α human IL-36β human IL-36γ Chimeric 1D9 33 48 (APE3798)HzD 1D9 6 39 0.035 0.033 0.027 (APE5281) Chimeric 5D3 34 49 (APE3849)Hzd 5D3 22 43 0.081 0.90 0.79 (APE6060) Hzd 5D3 22 44 0.088 0.117 0.078(APE6155) Hzd 5D3 24 44 0.09 0.105 0.084 (APE6194) Chimeric 18D4 35 506.0 3.0 (APE3847)

The results of this example demonstrate that antibodies composed ofcombinations of HCs and LCs described herein inhibit inflammatorycytokine release (IL-8) from human primary monocytes expressing IL-36Rand stimulated with cytokines IL-36α, IL-36β and IL-36γ in adose-dependent manner.

EXAMPLE 6

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides described herein can formantibodies that inhibit IL-36-dependent cytokine release from humanprimary peripheral blood mononuclear cells.

A Leukocyte Reduction System unit processed from a donor whole bloodunit was obtained from the San Diego Blood Bank. Peripheral bloodmononuclear cells (PBMCs) were prepped by standard methods using Ficolldensity centrifugation separation (Sigma HIS TOPAQUE™ cat #10771).

PBMCs were diluted to density of 1×10⁶ cells/ml in RPMI 1640 mediumcontaining 10% fetal bovine serum and penicillin/streptomycin, and 100μl cells per well were plated in standard flat-bottom 96-well tissueculture plates for a final cell density of 100,000 per well. Outsidewells were filled with 200 μl PBS per well to avoid edge effects. Platedcells were incubated for 2-3 hours in a 5% CO₂ 37° C. incubator to allowfor recovery.

After approximately 2-3 hours of culture, antibodies were added atconcentrations from 10 μg/ml or 1 μg/ml down to 0 by half-log dilutionsin culture medium. After 30 minutes, recombinant human IL-36 ligandswere added at approximately EC₅₀ concentrations (previously determinedempirically for each ligand) in culture medium. Antibody and ligandconcentrations were made at 4× of desired final concentrations and 50 μlper well were added for a final total volume in each well of 200 μl.Supernatants were removed approximately 48 hours later following athree-minute centrifugation of the plates, transferred to clean plates,and either tested immediately or stored at −80° C. until furtheranalysis.

Human IL-8 levels in the cell supernatants were assessed by ELISA usingR&D Systems DUO-SET™ ELISA kit (cat #DY208) following a standardprotocol provided by the manufacturer. Data were graphed and IC₅₀ valueswere calculated using GraphPad PRISM™ software.

The results of this assay are shown in FIGS. 7A-7C, and demonstrate thatantibodies composed of combinations of HCs and LCs described hereininhibit inflammatory cytokine release (IL-8) from human primaryperipheral blood mononuclear cells expressing IL-36R and stimulated withcytokines IL-36α, IL-36β and IL-36γ in a dose-dependent manner.

EXAMPLE 7

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides described herein can formantibodies that inhibit IL-36-dependent cytokine release from cynomolgusprimary peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMCs) were prepped by standardmethods using Ficoll density centrifugation separation (SigmaHISTOPAQUE™; cat #10771) from normal cynomolgus monkey whole bloodobtained from Biotox Sciences (San Diego, Calif.).

PBMCs were diluted to a density of 1×10⁶ cells/ml in RPMI 1640 mediumcontaining 10% fetal bovine serum and penicillin/streptomycin, and 100μl cells per well were plated in standard flat-bottom 96-well tissueculture plates for a final cell density of 100,000 per well. Outsidewells are filled with 200 μl PBS per well to avoid edge effects. Platedcells were incubated for 2-3 hours in a 5% CO₂ 37° C. incubator to allowfor recovery.

After approximately 2-3 hours of culture, antibodies were added atconcentrations from 10 μg/ml or 1 μg/ml down to 0 by half-log dilutionsin culture medium. After 30 minutes, recombinant cynomolgus IL-36ligands were added at approximately EC₅₀ concentrations (previouslydetermined empirically for each ligand) in culture medium. Antibody andligand concentrations were made at 4× of desired final concentrationsand 50 μl per well were added for a final total volume in each well of200 μl. Supernatants were removed approximately 48 hours later followinga three-minute centrifugation of the plates, transferred to cleanplates, and either tested immediately or stored at −80° C. until furtheranalysis.

Cynomolgus IL-8 levels in the cell supernatants were assessed by ELISAusing eBioscience monkey IL-8 platinum ELISA kit (San Diego, Calif.; cat#BMS640/3) following a standard protocol provided by the manufacturer.Data were graphed and IC₅₀ values were calculated using GraphPad PRISM™software. The results of this assay are set forth in Table 8.

TABLE 8 EC50 (nM) EC50 (nM) EC50 (nM) HC SEQ LC SEQ 50 ng/ml 10 ng/ml250 ng/ml Antibody ID NO: ID NO: cyno IL-36α cyno IL-36β cyno IL-36γ Hzd5D3 22 43 0.98 1.7 1.4 (APE6060) Hzd 5D3 22 44 1.2 1.5 0.89 (APE6155)Hzd 5D3 24 44 1.5 1.6 1.6 (APE6194)

The results of this example demonstrate that antibodies composed ofcombinations of HCs and LCs described herein inhibit inflammatorycytokine release (IL-8) from cynomolgus primary peripheral bloodmononuclear cells expressing IL-36R and stimulated with cytokinesIL-36α, IL-36β and IL-36γ in a dose-dependent manner.

EXAMPLE 8

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides described herein can formantibodies that cross-compete for binding to human IL-36R.

Cross-competition binding of the target IL-36R by antibodies comprisingvarious HC and LC polypeptides described herein was determined using aBIACORE™ T200 system (GE Healthcare, Little Chalfont, Buckinghamshire,UK). In each assay, the primary antibody was captured on the surface ofthe chip, and unutilized capture sites were subsequently blocked byaddition of saturating amounts of a negative control antibody which doesnot bind human IL-36R. This step was followed by binding of IL-36R andsubsequent addition of the secondary antibody to determine if theantibodies were competing for the same binding site on the monomericantigen. If antibodies bind the same epitope, no secondary binding wouldbe observed; if different binding sites on the IL-36R are utilized, thesecondary antibody would bind to the primary antibody/antigen complex.

Anti-human IgG (Fc-specific; GE Healthcare, Chalfont St. Giles, UnitedKingdom) was immobilized on the surface of a BIACORE™CMS chip at ˜8,000RU using EDC-activated coupling chemistry. Anti-IL-36R antibodiescomprising various combinations of the inventive HC and LC polypeptidesdescribed herein (10 μg/mL; 60s contact time at a flow rate of 10μL/min) were then captured on the surface of the chip at 25° C. yielding˜500RU captured antibody. The surface was blocked using a non-specific,isotype-matched negative control antibody to the target (APE4909 at 100μg/mL; 60 second contact time at a flow rate of 10 μL/min).Subsequently, IL-36R (at 1 μM) diluted in running buffer (HBS-EP+, pH7.6; GE Healthcare, Chalfont St. Giles, United Kingdom) was run over thesurface of the chip (300 seconds at a flow rate of 30 μL/min), and wasimmediately followed by a secondary antibody. The resulting sensogramsgenerated via surface plasmon resonance (SPR) indirectly monitoring masschanges on the surface of the chip were examined to determine crosscompetition between the antibodies.

The results of the competitive binding assays for the inventiveanti-IL-36R antibodies are shown in Table 9 and FIGS. 8A and 8B.

TABLE 9 Secondary Antibodies HC SEQ LC SEQ APE3847 APE5100 APE6155Primary Antibody ID NO: ID NO: (18D4) (1D9) (5D3) APE3847 (18D4) 35 50Competition No Competition competition APE5100 (1D9) 4 39 No CompetitionNo competition competition APE6155 (5D3) 22 44 Competition NoCompetition competition

The results of this example demonstrate that the antibodies APE6155(5D3) and APE3847 (18D4) compete for binding to the same epitope onhuman IL-36R, but do not compete with the antibody APE5100 for bindingto IL-36R, suggesting that neither APE6155 nor APE3847 shares an epitopewith APE5100. Competition results were consistent and independent of theordering of the primary and secondary antibody binding to antigen.

EXAMPLE 9

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides described herein can bind tocells expressing the human and cynomolgus monkey IL-36R with IL-1RAcP.

The binding of antibodies to CHO-K cells stably co-expressing humanIL-36R and human IL-1RAcP was examined. Cynomolgus IL-36R allelicvariation was examined by Sanger sequencing, and four distinct allelicvariants were identified within cynomolgus monkey populations. Thebinding of antibodies to CHO-K cells stably co-expressing cynomolgusmonkey IL-36R variant 1 and cynomolgus monkey IL-1RAcP was also examinedfor APE6155 and APE7247. Each antibody was incubated with CHO cellsharvested using accutase, washed, and seeded at 500,000 cells per well.Cells were incubated with antibodies at concentrations ranging from33nM-16 pM for 30 minutes at 4° C., and washed three times with FACSstaining buffer. Cells were spun and aspirated, and then the incubatedwith 100 μl paraformaldehyde for 10 minutes at room temperature. Cellswere again washed, aspirated, and stained with 100 μL of anti-human IgGAlexa 647 for 20 minutes at 4° C. Cells were resuspended in 100 μL FACSanalysis buffer before analysis on the FACS Array (BD Biosciences).

The results of the competitive binding assays for the inventiveanti-IL-36R antibodies are shown in FIGS. 9A and 9B. FIG. 9A showsbinding of APE06155 and APE07247 antibodies to CHO cells stablyexpressing human IL-36R and human IL-36R, and FIG. 9B shows the sameantibodies binding to CHO cells stably expressing cynomolgus monkeyvariant 1 IL-36R and IL-1RAcP. Data were fit using Graphpad Prismsoftware, with EC50 values for APE6155 determined as 1.5 nM and 2.4 nMto human and cynomolgus IL-36R expressing CHO cells respectively, and2.8 nM and 3.3 nM for backup Ab APE7247 binding to human and cynomolgusIL-36R expressing CHO cells, respectively. The negative isotype matchedcontrol antibody APE00422 showed no binding to either cell line.

EXAMPLE 10

This example demonstrates that the inventive immunoglobulin heavy chain(HC) and light chain (LC) polypeptides described herein can be used invivo in Cynomologous monkey with good pharmacokinetic characteristicsand subcutaneous bioavailability. Cynomologous monkeys were dosed withANB019 as a single dose intravenous (IV) or subcutaneous (SC) injection.Blood samples were collected from the monkeys in the single dose studyfrom 0.5 to 672 hrs (4 wks) after dosing. The derived serum samples wereanalyzed at AnaptysBio, Inc. (San Diego, Calif.) using an in-houseELISA-based method. Pharmacokinetic analyses were performed on the serumconcentration of ANA020 versus time data by AnaptysBio, Inc.

Serum concentration vs. time profiles of ANB019 behaved normally forboth dose routes, IV and SC, with levels dropping rapidly from Tmaxthrough the 24 hr time point for the IV administration, followed by adecline in line with the expected behavior of a monoclonal antibody in anonhuman primate. Pharmacokinetic parameter estimates from the ANB019serum concentration values were derived from a non-compartmentalanalysis and are listed in Table 10. Parameter estimates were consistentwith the anticipated pharmacokinetics for an IgG4 scaffold monoclonalantibody in the monkey. The half-life of ANB019 was estimated to be ˜270hrs after IV injection and ˜330 hrs after SC injection. Bioavailabilityafter SC injection was 60%.

TABLE 10 IgG4 IV IgG4 SC AUC_(0-672 hrs) (hr*ng/mL) 70,834,32542,680,650 T_(1/2) (hrs) 271 331 C_(max) (ng/mL) 757,588 149,518 T_(max)(hrs) 0.5 28 Bioavailability (%) 60

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An interleukin-36 receptor (IL-36R) bindingagent comprising an immunoglobulin heavy chain comprising CDR1, CDR2,and CDR3 of SEQ ID NO: 22, and an immunoglobulin light chain comprisingCDR1, CDR2, and CDR3 of SEQ ID NO: 44, wherein the IL-36R binding agentis an antibody or antigen-binding antibody fragment.
 2. The IL-36Rbinding agent of claim 1, wherein the IL-36R binding agent is part of anantibody conjugate.
 3. The IL-36R-binding agent of claim 1, wherein theIL-36R binding agent is a F(ab′)₂, Fab′, Fab, Fv, scFv, dsFv, or asingle chain binding polypeptide.
 4. The IL-36R-binding agent of claim1, wherein the IL-36R binding agent is an IgG1 antibody.
 5. The IL-36Rbinding agent of claim 1, wherein the IL-36R-binding agent binds toIL-36R with a K_(D) between about 1 picomolar (pM) and about 200 pM whenmeasured by surface plasmon resonance analysis.
 6. A pharmaceuticalcomposition comprising (a) the IL-36R-binding agent of claim 1, and (b)a pharmaceutically acceptable carrier.
 7. An anti-interleukin-36receptor (IL-36R) binding agent comprising the immunoglobulin heavychain variable region of SEQ ID NO: 22, and the immunoglobulin lightchain variable region of SEQ ID NO: 44, wherein the IL-36R binding agentis an antibody or antigen-binding antibody fragment.
 8. The IL-36Rbinding agent of claim 7, wherein the IL-36R binding agent is part of anantibody conjugate.
 9. The IL-36R-binding agent of claim 7, wherein theIL-36R binding agent is a F(ab′)₂, Fab′, Fab, Fv, scFv, dsFv, or asingle chain binding polypeptide.
 10. The IL-36R-binding agent of claim7, wherein the IL-36R binding agent is an IgG1 antibody.
 11. The IL-36Rbinding agent of claim 7, wherein the IL-36R-binding agent binds toIL-36R with a K_(D) between about 1 picomolar (pM) and about 200 pM whenmeasured by surface plasmon resonance analysis.
 12. A pharmaceuticalcomposition comprising (a) the IL-36R-binding agent of claim 7, and (b)a pharmaceutically acceptable carrier.
 13. A method of treating adisorder in a mammal that is responsive to IL-36R inhibition, whichmethod comprises administering an effective amount of the IL-6R bindingagent of claim 1 to a mammal with a disorder that is responsive toIL-36R inhibition, whereupon the disorder is treated.
 14. The method ofclaim 13, wherein the IL-36R binding agent is part of an antibodyconjugate.
 15. The method of claim 13, wherein the IL-36R binding agentis a F(ab′)₂, Fab′, Fab, Fv, scFv, dsFv, dAb, or a single chain bindingpolypeptide.
 16. The method of claim 13, wherein the half-life of theIL-36R-binding agent in the mammal is between 30 minutes and 45 days.17. The method of claim 13, wherein the IL-36R-binding agent binds toIL-36R with a K_(D) between about 1 picomolar (pM) and about 200 pM whenmeasured by surface plasmon resonance analysis.
 18. A method of treatinga disorder in a mammal that is responsive to IL-36R inhibition, whichmethod comprises administering an effective amount of the IL-36R bindingagent of claim 7 to a mammal with a disorder that is responsive toIL-36R inhibition, whereupon the disorder is treated.
 19. The method ofclaim 18, wherein the IL-36R binding agent is part of an antibodyconjugate.
 20. The method of claim 18, wherein the IL-36R binding agentis a F(ab′)₂, Fab′, Fab, Fv, scFv, dsFv, dAb, or a single chain bindingpolypeptide.
 21. The method of claim 18, wherein the half-life of theIL-36R-binding agent in the mammal is between 30 minutes and 45 days.22. The method of claim 18, wherein the IL-36R-binding agent binds toIL-36R with a K_(D) between about 1 picomolar (pM) and about 200 pM whenmeasured by surface plasmon resonance analysis.
 23. A nucleic acidencoding the immunoglobulin heavy chain and/or immunoglobulin lightchain of the IL-36R binding agent of claim 1, optionally in a vector.24. An isolated cell comprising the nucleic acid of claim
 23. 25. Amethod of preparing an IL-36R binding agent of claim 1, the methodcomprising expressing in a cell in vitro a nucleic acid sequenceencoding the immunoglobulin heavy chain of claim 1, and a nucleic acidsequence encoding the immunoglobulin light chain of claim
 1. 26. Anucleic acid encoding the immunoglobulin heavy chain and/orimmunoglobulin light chain of IL-36R binding agent of claim 7,optionally in a vector.
 27. An isolated cell comprising the nucleic acidof claim
 26. 28. A method of preparing an IL-36R binding agent of claim7, the method comprising expressing in a cell in vitro a nucleic acidsequence encoding the immunoglobulin heavy chain of claim 7, and anucleic acid sequence encoding the immunoglobulin light chain of claim7.